Multispecific antigen binding proteins

ABSTRACT

Multimeric multispecific proteins formed from dimerization between CH1 and CK domains and that bind two target antigens are provided. The proteins have advantages in production and in the treatment of disease, notably cancer or infectious disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.15/321,674, filed Dec. 22, 2016, now U.S. Pat. No. 11,208,480, which isa U.S. Nat'l Phase application of Int'l Appl. No. PCT/EP2015/064070,filed Jun. 23, 2015, which claims priority to U.S. Provisional Appl. No.62/017,913, filed Jun. 27, 2014, each of which is incorporated herein byreference.

REFERENCE TO THE SEQUENCE LISTING

This application includes as part of its disclosure an electronicsequence listing text file named “11562150001402.txt”, having a size of203,910 bytes and created on Nov. 17, 2021, which is hereby incorporatedby reference in its entirety.

FIELD OF THE INVENTION

Multispecific proteins that bind and specifically redirect NK cells tolyse a target cell of interest are provided. The proteins formats haveutility in the treatment of disease.

BACKGROUND

Bispecific antibodies binding two different epitopes and offeropportunities for increasing specificity, broadening potency, andutilizing novel mechanisms of action that cannot be achieved with atraditional monoclonal antibody. A variety of formats for bispecificantibodies that bind to two targets simultaneously have been reported.Cross-linking two different receptors using a bispecific antibody toinhibit a signaling pathway has shown utility in a number ofapplications (see, e.g., Jackman, et al., (2010) J. Biol. Chem.285:20850-20859). Bispecific antibodies have also been used toneutralize two different receptors. In other approaches, bispecificantibodies have been used to recruit immune effector cells, where T-cellactivation is achieved in proximity to tumor cells by the bispecificantibody which binds receptors simultaneously on the two different celltypes (see Baeuerle, P. A., et al, (2009) Cancer Res 69(12):4941-4).Most such approaches involve bispecific antibodies that link the CD3complex on T cells to a tumor-associated antigen. The most well-studiedbispecific antibody formats are “BiTe” antibodies and “DART’ antibodieswhich do not comprise Fe domains. However these antibodies are known tobe difficult to produce, require length cell development, have lowproductions yields and/or cannot be produced (based on publishedliterature) as a homogenous protein composition. In another example, abispecific antibody having one arm which bound FcγRIII and another whichbound to the HER2 receptor was developed for therapy of ovarian andbreast tumors that overexpress the HER2 antigen.

However, despite the existence of a variety of formats for bispecificantibodies, there is therefore a need in the art for proteins with newand well-defined mechanisms of action that can bind two or morebiological targets, and that have attractive properties for industrialdevelopment.

SUMMARY OF THE INVENTION

The present invention arises from the discovery of a functionalmultispecific antibody that permits a wide range of antibody variableregions to be readily used, having advantages in manufacturing by beingadapted to standard recombinant production techniques, and havingimproved in vivo pharmacology. The antibodies are particularly adaptedto bind a first antigen on a target cell to be eliminated and a secondantigen on an immune effector cell (e.g. an NK cell and/or a T cell),where the effector cells are directed to the target cell, e.g. a cancercell. The antigen on the effector cell can advantageously be anactivating receptor. When the multispecific antibody is designed to lackFcγR binding, it will not substantially activate effector cells viaCD16, and the multispecific antibody will be selective for theparticular effector cells of interest, as a function of the antigenbound by the multispecific antibody's hypervariable regions, therebyavoiding any unwanted FcγR/immune-mediated toxicity (e.g.cytokine-mediated toxicity) and/or inhibitory-FcγR mediated inhibitionof the effector cells targeted. The multispecific polypeptide iscapable, for example, of directing target antigen-expressing effectorcells to lyse a target cell expressing a target antigen, e.g. cancerantigen, viral antigen, etc. The multispecific antibody is particularlyeffective when binding both effector cell surface protein and a secondantigen (an antigen expressed by a target cell) in monovalent fashion.

In one embodiment, provided is a hetero-multimeric multispecific protein(e.g. a heterodimer, a heterotrimer) that binds a first and a secondantigen in monovalent fashion and that binds FcRn, the proteincomprising: a first antigen binding domain (ABD₁) that specificallybinds to a first antigen of interest, a second antigen binding domain(ABD₂) that specifically binds a second antigen of interest, and atleast a portion of a human Fc domain, e.g. an Fc domain that is bound byFcRn, optionally wherein the multispecific antibody is designed to havedecreased or substantially lack FcγR binding. In one embodiment, the Fcdomain is interposed between the two ABDs (one ABD is placed N-terminaland the other is C-terminal to the Fc domain).

In one embodiment, one of the antigens of interest is an activatingreceptor present on an effector cell, the other is a target cell antigen(e.g. a tumor antigen, a viral antigen, a microbial antigen), and themultispecific protein is bound by FcRn and has decreased orsubstantially lack FcγR binding, and the multi-specific protein can, inthe presence of the effector cells targeted and target cells, inducesignaling in and/or activation of the effector cells through theeffector cell polypeptide (the protein acts as an agonist), therebypromoting activation of the effector cells and/or lysis of target cellsin a directed manner. Notably, the multi-specific proteins can direct animmune effector response (cytotoxic response) toward a target cell thatis substantially limited to the effector cells of interest (the effectorcell receptor-expressing cells), and without activating FcγR-mediatedtoxicity or inhibitor FcγR-mediated inhibition. In one embodiment,provided is an isolated multimeric protein that binds monovalently to anactivating receptor expressed by an effector (e.g. T or NK cell) and ato cancer, viral or bacterial antigen, optionally wherein the protein isa hetero-dimeric or heterotrimeric protein, optionally wherein theprotein comprises at least two polypeptide chains formed by CH1-CKdimerization, the protein comprising: (a) a first antigen binding domainthat binds to an activating receptor expressed by an effector (e.g. T orNK cell) cell; (b) a second antigen binding domain that binds a cancer,viral or bacterial antigen expressed on a target cell; and (c) amonomeric or dimeric human Fc domain, wherein the protein is capable ofbinding via its Fc domain human neonatal Fc receptor (FcRn) and havingdecreased binding to a human Fcγ receptor compared to a full length wildtype human IgG1 antibody.

In one aspect of any embodiment herein, a multi-specific protein thatbinds an activating receptor on an effector cells can for example becharacterized by:

(a) ability to activate effector cells that express the activatingreceptor, when incubated with such effector cells in the presence oftarget cells; and/or

(b) lack of ability to activate such effector cells when incubated withsuch effector cells, in the absence of target cells. Optionally, theeffector cells are purified NK or T cells.

In one aspect of any embodiment herein, a multi-specific proteindescribed herein can for example be characterized by:

(a) ability to induce effector cells that express the activatingreceptor to lyse target cells, when incubated such effector cells in thepresence of target cells; and/or

(b) lack of ability to activate such effector cells when incubated withsuch effector cells, in the absence of target cells.

In one aspect of any embodiment herein, a multi-specific proteindescribed herein can for example be characterized by:

(a) ability to induce effector cells that express the activatingreceptor to lyse target cells, when incubated such effector cells in thepresence of target cells; and/or

(b) lack of ability to activate effector cells that express CD16 but donot express the activating receptor, when incubated with such effectorcells in the presence of target cells.

The multimeric polypeptide is composed of 2 or 3 different polypeptidechains in which 1 or 2 chains dimerize with a central chain based onCH1-CK heterodimerization (disulfide bond are formed between CH1 and CKdomains). The multimer may be composed of a central (first) polypeptidechain comprising two immunoglobulin variable domains that are part ofseparate antigen binding domains of different antigen specificities,with an Fc domain interposed between the two immunoglobulin variabledomains on the polypeptide chain, and a CH1 or CK constant domain placedon the polypeptide chain adjacent to one of, or each of, the variabledomain. Examples of the central polypeptide chain domain arrangementsare as follows, where each V₁, V₂ or V₃ is a variable domain:

V₁-(CH1 or CK)-Fc domain-V₂;

V₂-Fc domain-(CH1 or CK);

V₁-(CH1 or CK)-Fc domain-V₂-V₃;

V₂-V₃-Fc domain-V₁-(CH1 or CK); and

V₁-(CH1 or CK)-Fc domain-V₂-(CH1 or CK)

The Fc domain may be a full Fc domain or a portion thereof sufficient toconfer the desired functionality (e.g. FcRn binding). A secondadditional polypeptide chain will then be configured which will comprisean immunoglobulin variable domain and a CH1 or CK constant regionselected so as to permit CH1-CK heterodimerization with the centralpolypeptide chain; the immunoglobulin variable domain will be selectedso as to complement the variable domain of the central chain that isadjacent to the CH1 or CK domain, whereby the complementary variabledomains form an antigen binding domain for a first antigen of interest.

The antigen binding domain for the second antigen of interest can thenbe formed according to several configurations. In a first configuration,the central polypeptide chain comprises three immunoglobulin variabledomains, wherein the first variable domain is part of (together with theV domain in the second polypeptide) the antigen binding domain for afirst antigen of interest and the second and third variable domains areconfigured as tandem variable domains forming the antigen binding domainfor the second antigen of interest (e.g. a heavy chain variable domain(VH) and a light chain (kappa) variable domain (VK), for example formingan scFv unit).

In another configuration, a third polypeptide chain is provided so as toprovide the second variable region of the second antibody bindingdomain. Similarly to the second chain, the third chain will comprise animmunoglobulin variable domain and a CH1 or CK constant region selectedso as to permit CH1-CK heterodimerization with the central polypeptidechain. In this configuration the central chain will comprise two V-(CH1or CK) units with an interposed Fc domain, a first V-(CH1 or CK) unitwith form a CH1-CK heterodimer with a V-(CH1 or CK) unit of the secondchain, and the second V-(CH1 or CK) unit will form a heterodimer with aV-(CH1 or CK) unit of the third chain. The immunoglobulin variabledomain of the third chain will be selected so as to complement theunpaired variable domain of the central chain, whereby the complementaryvariable domains form an antigen binding domain for a second antigen ofinterest. Because CH1 and CK domains will form heterodimers so long asthe V domains adjacent thereto are complementary (i.e. not both VH orboth VK), one can select variable and constant domains that are notnaturally associated with one another so as to configure the variableand constant domains on the second and third chains such that eachV-(CH1 or CK) unit on the second and third chains finds a preferredbinding partner on the central chain. E.g. a VH-CK unit willheterodimerize with a VK-CH1 but not with a VK-CK. This will permit thepreferred pairing of the chains during production.

The multimeric polypeptide can be designed to have a monomeric Fc domainor a dimeric Fc domain. For monomeric Fc domains, the Fc domain maycomprise a CH3 domain having one or more amino acid mutations (e.g.substitutions) in the CH3 dimer interface to prevent CH3-CH3dimerization.

In one aspect, provided is a heterotrimeric bispecific antibody thatbinds a first and a second antigen of interest in monovalent fashion,wherein the antibody comprises a monomeric or dimeric Fc domain thatbinds human FcRn, optionally further wherein the Fc domain does not binda human Fcγ receptor.

In one embodiment, provided is a heteromultimeric, e.g. heterodimeric,bispecific polypeptide comprising: (a) a first polypeptide chaincomprising a first variable region (V), fused to a CH1 or CK domain,wherein the V-(CH1/CK) unit is in turn fused to a first terminus (N- orC-terminus) of a human Fc domain (a full Fc domain or a portionthereof); (b) a second polypeptide chain comprising a first variableregion (V) fused to a CH1 or CK domain that is complementary with theCH1 or CK of the first chain to form a CH1-CK dimer, optionally whereinthe V-(CH1/CK) unit is fused to at least a human Fc domain (a full Fcdomain or a portion thereof), wherein the two first variable regionsform an antigen binding domain that binds a first antigen of interest inmonovalent fashion, and (c) an antigen binding domain that binds asecond antigen (optionally together with a complementary antigen bindingdomain), fused to a second terminus (N- or C-terminus) of the Fc domainof the first polypeptide (or of the second Fc-derived polypeptide, ifsuch polypeptide comprises an Fc domain) such that the Fc domain isinterposed between the V-(CH1/CK) unit and the antigen binding domainthat binds a second antigen. Optionally the first and second polypeptidechains are bound by interchain disulfide bonds, e.g. formed betweenrespective CH1 and CK domains. Optionally a V-(CH1/CK) unit is fused toa human Fc domain directly, or via intervening sequences, e.g. hingeregion, linker, other protein domain(s), etc.

In one embodiment of the above heteromultimeric polypeptide, thepolypeptide is a heterodimer, wherein the antigen binding domain for asecond antigen is an scFv, optionally an scFv that binds an activatingreceptor on an effector cell.

In one aspect provided is an isolated hetero-multimeric polypeptide thatbinds a first and second antigen of interest in monovalent fashion,comprising:

-   -   (a) a first polypeptide chain comprising a first variable        domain (V) fused to a CH1 of CK constant region, a second        variable domain, and an Fc domain or portion thereof interposed        between the first and second variable domains; and    -   (b) a second polypeptide chain comprising a first variable        domain (V) fused at its C-terminus to a CH1 or CK constant        region selected to be complementary to the CH1 or CK constant        region of the first polypeptide chain such that the first and        second polypeptides form a CH1-CK heterodimer in which the first        variable domain of the first polypeptide chain and the first        variable domain of the second polypeptide form an antigen        binding domain that binds the first antigen of interest.

Optionally, the first polypeptide chain comprises a first variabledomain (V) fused at its C-terminus to a CH1 and the second polypeptidechain comprises a first variable domain fused at its C-terminus to a CKconstant region, such that the first and second polypeptides form aCH1-CK heterodimer. Alternatively, the first polypeptide chain comprisesa first variable domain (V) fused at its C-terminus to a CK and thesecond polypeptide chain comprises a first variable domain fused at itsC-terminus to a CH1 constant region, such that the first and secondpolypeptides form a CK-CH1 heterodimer. Optionally, the first variabledomain of the first polypeptide chain and the first variable domain ofthe second polypeptide chain are derived from the same first parentalantibody that specifically binds the first antigen, and the secondvariable domain of the first polypeptide is from a second parentalantibody that specifically binds the second antigen.

In one embodiment, the first polypeptide chain further comprises a thirdvariable domain fused to the second variable domain,

wherein the first and second polypeptide form a CH1-CK heterodimer,wherein the first variable domain of the first polypeptide chain and thefirst variable domain of the second polypeptide chain form an antigenbinding domain specific for the first antigen of interest, and whereinthe second and third variable domains of the first polypeptide chainform an scFv specific for the second antigen of interest. Optionallyfirst polypeptide chain has the domain arrangement: V₂-V₃-Fcdomain-V₁-(CH1 or CK), such that a hetero-multimeric polypeptide isformed having the domain arrangement:

wherein one of V₁ of the first polypeptide chain and V₁ of the secondpolypeptide chain is a light chain variable domain and the other is aheavy chain variable domain.

Optionally first polypeptide chain has the domain arrangement: V₁-(CH1or CK)-Fc domain-V₂-V₃, such that a hetero-multimeric polypeptide isformed having the domain arrangement:

wherein one of V₁ of the first polypeptide chain and V₁ of the secondpolypeptide chain is a light chain variable domain and the other is aheavy chain variable domain.

In one aspect provided is an isolated heterodimeric polypeptide thatbinds a first and second antigen of interest in monovalent fashion,optionally wherein one of the antigens is expressed on an immuneeffector cell and the other is an antigen of interest, comprising:

(a) a first polypeptide chain comprising, from N- to C-terminus, a firstvariable domain

-   -   (V), a CH1 of CK constant region, a Fc domain or portion        thereof, a second variable domain and third variable domain; and

(b) a second polypeptide chain comprising, from N- to C-terminus, afirst variable domain (V), a CH1 or CK constant region, and optionally aFc domain or portion thereof, wherein the CH1 or CK constant region isselected to be complementary to the CH1 or CK constant region of thefirst polypeptide chain such that the first and second polypeptides forma CH1-CK heterodimer in which the first variable domain of the firstpolypeptide chain and the first variable domain of the secondpolypeptide form an antigen binding domain that binds the first antigenof interest; and wherein a second variable domain and third variabledomain forms an antigen binding domain that binds the second antigen ofinterest. When the second polypeptide chain lacks an Fc domain, thefirst polypeptide chain will comprise an Fc domain modified to preventCH3-CH3 dimerization (e.g., substitutions or tandem CH3 domain).

In one aspect provided is an isolated heterodimeric polypeptide thatbinds a first and second antigen of interest in monovalent fashion,optionally wherein one of the antigens is expressed on an immuneeffector cell and the other is an antigen of interest, comprising:

(a) a first polypeptide chain comprising, from N- to C-terminus, asecond variable domain and third variable domain, a Fc domain or portionthereof, a first variable domain (V), and a CH1 of CK constant region;and

(b) a second polypeptide chain comprising, from N- to C-terminus, afirst variable domain (V), a CH1 or CK constant region, wherein the CH1or CK constant region is selected to be complementary to the CH1 or CKconstant region of the first polypeptide chain such that the first andsecond polypeptides form a CH1-CK heterodimer in which the firstvariable domain of the first polypeptide chain and the first variabledomain of the second polypeptide form an antigen binding domain thatbinds the first antigen of interest; and

wherein a second variable domain and third variable domain forms anantigen binding domain that binds the second antigen of interest. Thefirst polypeptide chain can comprise an Fc domain modified to preventCH3-CH3 dimerization (e.g., substitutions or tandem CH3 domain).

In one embodiment, provided is a trimeric polypeptide, comprising:

-   -   (a) a first polypeptide chain comprising a first variable        domain (V) fused to a first CH1 or CK constant region, a second        variable domain (V) fused to a second CH1 or CK constant region,        and an Fc domain or portion thereof interposed between the first        and second variable domains (i.e. the Fc domain is interposed        between the first and second V-(CH1/CK) units);    -   (b) a second polypeptide chain comprising a variable domain        fused at its C-terminus to a CH1 or CK constant region selected        to be complementary to the first CH1 or CK constant region of        the first polypeptide chain such that the first and second        polypeptides form a CH1-CK heterodimer; and    -   (c) a third polypeptide chain comprising a variable domain fused        at its C-terminus to a CH1 or CK constant region, wherein the        variable domain and CH1 or CK constant region are selected to be        complementary to the second variable domain and CH1 or CK        constant region of the first polypeptide chain such that the        first and third polypeptides form a CH1-CK heterodimer bound by        disulfide bond(s) formed between the CH1 or CK constant region        of the third polypeptide and the second CH1 or CK constant        region of the first polypeptide, but not between the CH1 or CK        constant region of the third polypeptide and the first CH1 or CK        constant region of the first polypeptide

wherein the first, second and third polypeptides form a CH1-CKheterotrimer, and wherein the first variable domain of the firstpolypeptide chain and the variable domain of the second polypeptidechain form an antigen binding domain specific for a first antigen ofinterest, and the second variable domain of the first polypeptide chainand the variable domain on the third polypeptide chain form an antigenbinding domain specific for a second antigen of interest. Optionally,the first variable domain of the first polypeptide chain and thevariable domain of the second polypeptide chain are derived from thesame first parental antibody that specifically binds the first antigen,and the second variable domain of the first polypeptide and the variabledomain of the third polypeptide chain are derived from the same secondparental antibody that specifically binds the second antigen.

In one embodiment, provided is a trimeric polypeptide that binds a firstand second antigen of interest in monovalent fashion, optionally whereinone of the antigens is expressed on an immune effector cell and theother is an antigen of interest, comprising:

(a) a first polypeptide chain comprising, from N- to C-terminus, a firstvariable domain

-   -   (V) fused to a first CH1 or CK constant region, an Fc domain or        portion thereof, and a second variable domain (V) fused to a        second CH1 or CK constant region;

(b) a second polypeptide chain comprising, from N- to C-terminus, avariable domain fused to a CH1 or CK constant region selected to becomplementary to the first (but not the second) CH1 or CK constantregion of the first polypeptide chain such that the first and secondpolypeptides form a CH1-CK heterodimer, and optionally an Fc domain orportion thereof; and

(c) a third polypeptide chain comprising, from N- to C-terminus, avariable domain fused to a CH1 or CK constant region, wherein thevariable domain and CH1 or CK constant region are selected to becomplementary to the second (but not the first) variable domain and CH1or CK constant region of the first polypeptide chain. The first andthird polypeptides will therefore form a CH1-CK heterodimer formedbetween the CH1 or CK constant region of the third polypeptide and thesecond CH1 or CK constant region of the first polypeptide, but notbetween the CH1 or CK constant region of the third polypeptide and thefirst CH1 or CK constant region of the first polypeptide. The first,second and third polypeptides form a CH1-CK heterotrimer, and whereinthe first variable domain of the first polypeptide chain and thevariable domain of the second polypeptide chain form an antigen bindingdomain specific for a first antigen of interest, and the second variabledomain of the first polypeptide chain and the variable domain on thethird polypeptide chain form an antigen binding domain specific for asecond antigen of interest.

Provided also is a purified or homogenous composition, wherein at least90%, 95% or 99% of the proteins in the composition are a multimericpolypeptide of the disclosure.

Optionally, in any embodiment where two Fc domains are present in themultimeric polypeptide, an Fc domain comprises a CH2 and a CH3 domaincapable of CH3-CH3 dimerization.

Optionally in any embodiment, each of the variable domains is a singleimmunoglobulin heavy or light chain variable domain.

Optionally in any embodiment, a Fc domain is fused to an antigen bindingdomain, CH1 domain and/or CK domain via a hinge region or linkerpeptide.

Optionally in any embodiment, an Fc domain comprises a CH2 domain.Optionally, a CH2 domain is fused to an antigen binding domain, CH1domain and/or CK domain via a hinge region or linker peptide.Optionally, a CH2 domain comprises an amino acid substitution to reducebinding to a human Fcγ receptor. In one embodiment, the multispecificpolypeptide lacks N-linked glycosylation or has modified N-linkedglycosylation. In one embodiment, the multispecific polypeptidecomprises an N297X mutation, wherein X is any amino acid other thanasparagine.

Optionally in any embodiment, two polypeptide chains within a multimericpolypeptide are bound to one another by interchain disulfide bond(s)formed between respective hinge regions and/or respective CH1/CKconstant regions.

Optionally in any embodiment, the multispecific polypeptide (or the Fcportion thereof) is capable of binding to human neonatal Fc receptor(FcRn).

Optionally in any embodiment, the multispecific polypeptide (or the Fcportion thereof) has decreased binding to a human Fcγ receptor comparedto a full length wild type human IgG1 antibody. Optionally, themultispecific polypeptide (or the Fc portion thereof) substantiallylacks binding to a human Fcγ receptor.

In one aspect of any embodiment herein, the multimeric polypeptide(and/or its Fc domain) has decreased binding to a human Fcγ receptor(e.g. CD16, CD32A, CD32B and/or CD64). e.g., compared to a full lengthwild type human IgG1 antibody.

In one embodiment, the multimeric polypeptide has decreased (e.g.partial or complete loss of) antibody dependent cytotoxicity (ADCC),complement dependent cytotoxicity (CDC), antibody dependent cellularphagocytosis (ADCP), FcR-mediated cellular activation (e.g. cytokinerelease through FcR cross-linking), and/or FcR-mediated plateletactivation/depletion, as mediated by immune cells that do not express anantigen of interest bound by the variable regions of the multimericpolypeptide (i.e. in the absence of cells that express an antigen ofinterest bound by the variable regions), compared, e.g., to the samepolypeptide having a wild-type Fc domain of human IgG1 isotype.

In one aspect of any embodiment herein, the CH2 domain comprises anamino acid modification that decreases binding to a human Fcγ receptor,compared to a wild-type CH2 domain. In one embodiment the CH2-modifiedmultispecific polypeptide has decreased (e.g. partial or complete lossof) antibody dependent cytotoxicity (ADCC), complement dependentcytotoxicity (CDC), antibody dependent cellular phagocytosis (ADCP),FcR-mediated cellular activation (e.g. cytokine release through FcRcross-linking), and/or FcR-mediated platelet activation/depletion,mediated by immune effector cells that do not express antigen ofinterest bound by the multimeric polypeptide's ABDs, compared, e.g., tothe same polypeptide having a wild-type CH2 domain.

Optionally in any embodiment, each antigen binding domain comprises thehypervariable regions, optionally the heavy and light chain CDRs, of anantibody.

Optionally, in any embodiment where a single Fc domain is present in themultimeric polypeptide, an Fc domain(s) comprises a CH3 domaincomprising an amino acid substitution at 1, 2, 3, 4, 5, 6 or 7 of thepositions L351, T366, L368, P395, F405, T407 and/or K409 (EU numberingas in Kabat), or a tandem CH3 domain.

Optionally in any embodiment, the Fc domain(s) is a human IgG4 Fc domainor a portion thereof, optionally comprising one or more amino acidmodifications.

Optionally in any embodiment, one of the first or second antigen ofinterest is a cancer antigen and the other is a polypeptide expressed onthe surface of an immune effector cell.

Optionally in any embodiment, one of the first or second antigen ofinterest is a viral or bacterial antigen and the is a polypeptideexpressed on the surface of an immune effector cell.

Optionally in any embodiment, a variable domain comprises frameworkresidues from a human framework region, e.g., a variable domaincomprises 1, 2 or 3 CDRs of human or non-human origin and frameworkresidues of human origin.

In one aspect of any of the embodiments herein, the bispecificpolypeptide has a great binding affinity (monovalent) for a cancerantigen (or a viral or bacterial antigen) than for an antigen expressedby an immune effector cell. Such antibodies will provide foradvantageous pharmacological properties. In one aspect of any of theembodiments of the invention, the polypeptide has a Kd for binding(monovalent) to an antigen expressed by immune effector cell of lessthan 10⁻⁷ M, preferably less than 10⁻⁸ M, or preferably less than 10⁻⁹ Mfor binding to an polypeptide expressed by an immune effector cell;optionally the polypeptide has a Kd for binding (monovalent) to acancer, viral or bacterial antigen that is less than (i.e. has betterbinding affinity than) the Kd for binding (monovalent) to the antigenexpressed by immune effector cell.

In one embodiment of any of the polypeptides herein, the multispecificpolypeptide is capable of directing effector cells (e.g. a T cell, an NKcell) expressing one of first or second antigen of interest to lyse atarget cell expressing the other of said first of second antigen ofinterest (e.g. a cancer cell).

In one aspect of any of the embodiments herein, provided is arecombinant nucleic acid encoding a first polypeptide chain, and/or asecond polypeptide chain and/or a third polypeptide chain of any of theproteins of the disclosure. In one aspect of any of the embodimentsherein, provided is a recombinant host cell comprising a nucleic acidencoding a first polypeptide chain, and/or a second polypeptide chainand/or a third polypeptide chain of any of the proteins of thedisclosure, optionally wherein the host cell produces a protein of thedisclosure with a yield (final productivity, following purification) ofat least 1, 2, 3 or 4 mg/L. Also provided is a kit or set of nucleicacids comprising a recombinant nucleic acid encoding a first polypeptidechain of the disclosure, a recombinant nucleic acid encoding a secondpolypeptide chain of the disclosure, and, optionally, a recombinantnucleic acid encoding a third polypeptide chain of the disclosure. Alsoprovided are methods of making monomeric, heterodimeric andheterotrimeric proteins of the disclosure.

In one embodiment, the invention provides methods of making aheterodimeric protein (e.g. any heterodimeric protein described herein),comprising:

a) providing a first nucleic acid encoding a first polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a first variabledomain (V) fused to a CH1 of CK constant region, a second variabledomain (and optionally third variable domain, wherein the second andthird variable domain form an antigen binding domain), and an Fc domainor portion thereof interposed between the first and second variabledomains);

b) providing a second nucleic acid encoding a second polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a first variabledomain (V) fused at its C-terminus to a CH1 or CK constant regionselected to be complementary to the CH1 or CK constant region of thefirst polypeptide chain such that the first and second polypeptides forma CH1-CK heterodimer in which the first variable domain of the firstpolypeptide chain and the first variable domain of the secondpolypeptide form an antigen binding domain); wherein one of the first orsecond antigen binding domains binds a polypeptide on the surface of animmune effector cell and the other binds a tumor, viral or bacterialantigen; and

c) expressing said first and second nucleic acids in a host cell toproduce a protein comprising said first and second polypeptide chains,respectively; and recovering a heterodimeric protein. Optionally, theheterodimeric protein produced represents at least 20%, 25% or 30% ofthe total protein (e.g. bispecific proteins) obtained prior topurification. Optionally step (c) comprises loading the protein producedonto an affinity purification support, optionally an affinity exchangecolumn, optionally a Protein-A support or column, and collecting theheterodimeric protein; and/or loading the protein produced (e.g., theprotein collected following loading onto an affinity exchange or ProteinA column) onto an ion exchange column; and collecting the heterodimericfraction. In one embodiment, the second variable domain (optionallytogether with the third variable domain) of the first polypeptide chainbinds a polypeptide on the surface of an immune effector cell.

By virtue of their ability to be produced in standard cell lines andstandardized methods with high yields, unlike BITE, DART and otherbispecific formats, the proteins of the disclosure also provide aconvenient tool for screening for the most effective variable regions toincorporated into a multispecific protein. In one aspect, the presentdisclosure provides a method for identifying or evaluating candidatevariable regions for use in a heterodimeric protein, comprising thesteps of:

a) providing a plurality of nucleic acid pairs, wherein each pairincludes one nucleic acid encoding a heavy chain candidate variableregion and one nucleic acid encoding a light chain candidate variableregion, for each of a plurality of heavy and light chain variable regionpairs (e.g., obtained from different antibodies binding the same ordifferent antigen(s) of interest);

b) for each of the plurality nucleic acid pairs, making a heterodimericprotein by:

-   -   (i) producing a first nucleic acid encoding a first polypeptide        chain comprising one of the heavy or light chain candidate        variable domains (V) fused to a CH1 or CK constant region, a        second variable domain (and optionally third variable domain,        wherein the second and third variable domain form a first        antigen binding domain), and an Fc domain or portion thereof        interposed between the candidate and second variable domains);    -   (ii) producing a second nucleic acid encoding a second        polypeptide chain comprising the other of the heavy or light        chain candidate variable domains (V) fused at its C-terminus to        a CH1 or CK constant region selected to be complementary to the        CH1 or CK constant region of the first polypeptide chain such        that the first and second polypeptides form a CH1-CK heterodimer        in which the heavy and light chain candidate variable domains        form a second antigen binding domain; and    -   (iii) expressing said nucleic acids encoding the first and        second polypeptide chains in a host cell to produce a protein        comprising said first and second polypeptide chains,        respectively; and recovering a heterodimeric protein; and

c) evaluating the plurality of heterodimeric proteins produced for abiological activity of interest, e.g., an activity disclosed herein. Inone embodiment, the first antigen binding domain binds a polypeptide onthe surface of an immune effector cell and the second antigen bindingdomain a tumor, viral or bacterial antigen; optionally the first antigenbinding domain is an scFv. Optionally, the heterodimeric proteinproduced represents at least 20%, 25% or 30% of the total proteinobtained prior to purification. Optionally the recovering step in (iii)comprises loading the protein produced onto an affinity purificationsupport, optionally an affinity exchange column, optionally a Protein-Asupport or column, and collecting the heterodimeric protein; and/orloading the protein produced (e.g., the protein collected followingloading onto an affinity exchange or Protein A column) onto an ionexchange column; and collecting the heterodimeric fraction.

In one embodiment, provided is a method of making a heterotrimericprotein (e.g. any heterotrimeric protein described herein), comprising:

(a) providing a first nucleic acid encoding a first polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a first variabledomain (V) fused to a first CH1 or CK constant region, a second variabledomain fused to a second CH1 or CK constant region, and an Fc domain orportion thereof interposed between the first and second (V-CH1/CK)units);

(b) providing a second nucleic acid encoding a second polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a variable domain(V) fused at its C-terminus to a CH1 or CK constant region selected tobe complementary to the first CH1 or CK constant region of the firstpolypeptide chain such that the first and second polypeptides form aCH1-CK heterodimer in which the first variable domain of the firstpolypeptide chain and the variable domain of the second polypeptide forman antigen binding domain);

(c) providing a third nucleic acid comprising a third polypeptide chaindescribed herein (e.g., a polypeptide chain comprising a variable domainfused at its C-terminus to a CH1 or CK constant region, wherein the CH1or CK constant region is selected to be complementary to the secondvariable domain and second CH1 or CK constant region of the firstpolypeptide chain such that the first and third polypeptides form aCH1-CK heterodimer in which the second variable domain of the firstpolypeptide and the variable domain of the third polypeptide form anantigen binding domain; and

(d) expressing said first, second and third nucleic acids in a host cellto produce a protein comprising said first, second and third polypeptidechains, respectively; and recovering a heterotrimeric protein.Optionally, the heterotrimeric protein produced represents at least 20%,25% or 30% of the total protein obtained prior to purification.Optionally step (d) comprises loading the protein produced onto anaffinity purification support, optionally an affinity exchange column,optionally a Protein-A support or column, and collecting theheterotrimeric protein; and/or loading the protein produced (e.g., theprotein collected following loading onto an affinity exchange or ProteinA column) onto an ion exchange column; and collecting the heterotrimericfraction. Optionally, one of the antigen binding domains binds apolypeptide on the surface of an immune effector cell, and the otherbinds an antigen of interest. In one embodiment, the second or the thirdpolypeptide further comprises and Fc domain or fragment thereof fused tothe C-terminus of the CH1 or CK domain (e.g. via a hinge domain orlinker). In one embodiment, the second variable domain of the firstpolypeptide and the variable domain of the third polypeptide form anantigen binding domain that binds a polypeptide on the surface of animmune effector cell.

In one aspect, the present disclosure provides a method for identifyingor evaluating candidate variable regions for use in a heterotrimericprotein, comprising the steps of:

a) providing a plurality of nucleic acid pairs, wherein each pairincludes one nucleic acid encoding a heavy chain candidate variableregion and one nucleic acid encoding a light chain candidate variableregion, for each of a plurality of heavy and light chain variable regionpairs (e.g., obtained from different antibodies binding the same ordifferent antigen(s) of interest);

b) for each of the plurality nucleic acid pairs, making a heterotrimericprotein by:

-   -   (i) producing a first nucleic acid encoding a first polypeptide        chain comprising one of the heavy or light chain candidate        variable domains (V) fused to a first CH1 or CK constant region,        a second variable domain fused to a second CH1 or CK constant        region, and an Fc domain or portion thereof interposed between        the first and second (V-CH1/CK) units);    -   (ii) producing a second nucleic acid encoding a second        polypeptide chain comprising the other of the heavy or light        chain candidate variable domains (V) fused at its C-terminus to        a CH1 or CK constant region selected to be complementary to the        first CH1 or CK constant region of the first polypeptide chain        such that the first and second polypeptides form a CH1-CK        heterodimer in which the heavy and light chain candidate        variable domains form an antigen binding domain;    -   (ii) producing a third nucleic acid encoding a third polypeptide        chain comprising a variable domain fused at its C-terminus to a        CH1 or CK constant region, wherein the CH1 or CK constant region        is selected to be complementary to the second variable domain        and second CH1 or CK constant region of the first polypeptide        chain such that the first and third polypeptides form a CH1-CK        heterodimer in which the second variable domain of the first        polypeptide and the variable domain of the third polypeptide        form an antigen binding domain; and    -   (iii) expressing said nucleic acids encoding the first and        second polypeptide chains in a host cell to produce said first        and second polypeptide chains, respectively; and recovering a        heterodimeric protein; and

c) evaluating the plurality of heterodimeric proteins produced for abiological activity of interest, e.g., an activity disclosed herein. Inone embodiment, the second or the third polypeptide further comprisesand Fc domain or fragment thereof fused to the C-terminus of the CH1 orCK domain (e.g. via a hinge domain or linker). Optionally, theheterotrimeric protein produced represents at least 20%, 25% or 30% ofthe total proteins obtained prior to purification. Optionally therecovering step in (iii) comprises loading the protein produced onto anaffinity purification support, optionally an affinity exchange column,optionally a Protein-A support or column, and collecting theheterotrimeric protein; and/or loading the protein produced (e.g., theprotein collected following loading onto an affinity exchange or ProteinA column) onto an ion exchange column; and collecting the heterotrimericfraction.

In the methods for identifying or evaluating candidate variable regions,candidate variable regions can for example be from antibodies that bindsa polypeptide on the surface of an immune effector cell, or fromantibodies that bind an antigen of interest, e.g. a tumor, bacterial orviral antigen. When the candidate variable regions are from antibodiesagainst a tumor, bacterial or viral antigen, the other variable regioncan be from an antibody that binds a polypeptide on the surface of animmune effector cell, which will permit a panel of antibodies to thetumor, bacterial or viral antigen to be tested in the context of ananti-effector cell ABD which has been determined to be effective. Itwill also be appreciated that the position of the respective ABDs forthe candidate variable region pair and the other variable region paircan be inverted. For example, in a trimeric protein the methods can bemodified such that the heavy and light chain candidate variable domainsare formed by the second V region of the first polypeptide and the Vregion of the second polypeptide, and the other variable region pair areformed by the first V region of the first polypeptide and the V regionof the third polypeptide.

In one aspect, the present disclosure provides a method for identifyingor evaluating candidate protein configurations for use in ahetero-multimeric protein, comprising the steps of:

producing, separately (e.g. in separate containers), a plurality ofhetero-multimeric proteins of the disclosure, wherein the proteinsdiffer in their domain arrangements, and

evaluating the plurality of hetero-multimeric proteins produced for abiological activity of interest, e.g., an activity disclosed herein. Inone embodiment, the proteins having different domain arrangements shareantigen binding domains (e.g. the same CDRs or variable domains) for thefirst and/or second antigen of interest. In one embodiment 2, 3, 4, 5,6, 7 or more different proteins are produced and evaluated. In oneembodiment, one or more of (or all of) the proteins are selected fromthe group of proteins having a domain arrangement disclosed herein, e.g.that of formats F2, F5, F6, F7, F8, F9, F10, F11, F12, F13, F14, F15,F16 and F17. In one embodiment the proteins are produced according tothe methods disclosed herein.

In one aspect, the present disclosure provides a library of at least 5,10, 20, 30, 50 hetero-multimeric proteins of the disclosure, wherein theproteins share domain arrangements but differ in the amino acid sequenceof the variable domain of one or both of their antigen binding domains.

In one aspect, the present disclosure provides a library of at least 2,3, 4, 5 or 10 hetero-multimeric proteins of the disclosure, wherein theproteins share the amino acid sequence of the variable domain of one orboth of their antigen binding domains, but differ in domainarrangements.

In one embodiment, the second variable domain of the first polypeptideand the variable domain of the third polypeptide form an antigen bindingdomain that binds an activating receptor on an effector cell. In oneembodiment, evaluating heterodimeric or heterotrimeric proteins for acharacteristic of interest comprises evaluating the proteins for one ormore properties selected from the group consisting of: binding to anantigen of interest, binding to an FcRn receptor, binding to an Fcγreceptor, Fc-domain mediated effector function(s), agonistic orantagonistic activity at a polypeptide to which the multimeric proteinsbinds, ability to modulate the activity (e.g. cause the death of) a cellexpressing the antigen of interest, ability to direct a lymphocyte to acell expressing the antigen of interest, ability to activate alymphocyte in the presence and/or absence of a cell expressing theantigen of interest, lymphocyte (e.g. T cell or NK cell) activation inpresence but not in absence of target cells, lack of activation ofantigen-of-interest-negative lymphocytes, stability or half-life invitro or in vivo, production yield, purity within a composition, andsusceptibility to aggregate in solution.

In one aspect, the present disclosure provides a method for identifyingor evaluating a hetero-multimeric protein, comprising the steps of:

(a) providing nucleic acids encoding a hetero-multimeric proteindescribed herein;

(b) expressing said nucleic acids in a host cell to produce saidprotein, respectively; and recovering said protein; and

(c) evaluating the protein produced for a biological activity ofinterest, e.g., an activity disclosed herein. In one embodiment, aplurality of different proteins are produced and evaluated.

In one embodiment, the protein binds an activating receptor on aneffector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress the activating receptor, when incubated with such effector cellsin the presence of target cells (that express antigen of interest).Optionally, step (i) is followed by a step comprising: selecting aprotein (e.g., for further development, for use as a medicament) thatactivates said effector cells.

In one embodiment, the protein binds an activating receptor on aneffector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress the activating receptor, when incubated with such effector cellsin the absence of target cells (that express antigen of interest).Optionally, step (i) is followed by a step comprising: selecting aprotein (e.g., for further development, for use as a medicament) thatdoes not substantially activate said effector cells.

In one embodiment, the protein binds an activating receptor on aneffector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress the activating receptor, when incubated with such effector cellsin the presence of target cells (that express antigen of interest); and

(ii) testing the ability of the protein to activate effector cells thatexpress the activating receptor, when incubated with such effector cellsin the absence of target cells (that express antigen of interest).Optionally, the method further comprises: selecting a protein (e.g., forfurther development, for use as a medicament) that does notsubstantially activate said effector cells when incubated in the absenceof target cells, and that activates said effector cells when incubatedin the presence of target cells.

In one embodiment, the protein binds an activating receptor on aneffector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the polypeptide to induce effector cells thatexpress the activating receptor to lyse target cells (that expressantigen of interest), when incubated such effector cells in the presenceof target cells. Optionally, step (i) is followed by a step comprising:selecting a protein (e.g., for further development, for use as amedicament) that induces effector cells that express the activatingreceptor to lyse the target cells, when incubated such effector cells inthe presence of the target cells.

In one embodiment, the protein binds an activating receptor on aneffector cell and an antigen of interest, and the step (c) comprises:

(i) testing the ability of the protein to activate effector cells thatexpress CD16 but do not express the activating receptor, when incubatedwith such effector cells in the presence of target cells. Optionally,step (i) is followed by a step comprising: selecting a protein (e.g.,for further development, for use as a medicament) that do notsubstantially activate said effector cells, when incubated with sucheffector cells in the presence of target cells.

In one aspect provided is a pharmaceutical composition comprising acompound or composition described herein, and a pharmaceuticallyacceptable carrier.

In one aspect provided is the use of a polypeptide or composition of anyone of the above claims as a medicament for the treatment of disease.

In one aspect provided is a method of treating a disease in a subjectcomprising administering to the subject a compound or compositiondescribed herein.

In one embodiment, the disease is a cancer or an infectious disease.

Any of the methods can further be characterized as comprising any stepdescribed in the application, including notably in the “DetailedDescription of the Invention”). The invention further relates to aprotein obtainable by any of present methods. The disclosure furtherrelates to pharmaceutical or diagnostic formulations of the antibodiesof the present invention. The disclosure further relates to methods ofusing antibodies in methods of treatment or diagnosis.

These and additional advantageous aspects and features of the inventionmay be further described elsewhere herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows two examples of multispecific polypeptides in which one ofthe antigen binding domains (ABD₁ or ABD₂) specifically binds to NKp46and the other of the ABDs binds to an antigen of interest, wherein thedrawing on the left has tandem scFv and the drawing on the right has twoABD with an Fc domain interposed.

FIG. 2 shows a schematic of an anti-CD19-F1-Anti-NKp46 used in theExamples herein. The star in the CH2 domain indicates an option N297Smutation.

FIG. 3 shows a schematic of an anti-CD19-Anti-NKp46-IgG1-Fcmono. For thescFv tandem construct, the Anti-NKp46 VK domain (C-terminal) is linkedto the CH2 domain (N-terminal) using a linker peptide (RTVA) that mimicsthe regular VK-CK elbow junction.

FIG. 4 shows that Anti-CD19-F1-Anti-CD3 does not cause T/B cellaggregation in the presence of B221 (CD19) or JURKAT (CD3) cell lineswhen separate, but it does cause aggregation of cells when both B221 andJURKAT cells are co-incubated.

FIG. 5 shows Anti-CD19-F1-Anti-CD3 retains binding to FcRn, with a 1:1ratio (1 FcRn for each monomeric Fc) (KD=194 nM), in comparison to achimeric full length antibody having human IgG1 constant regions(KD=15.4 nM) which binds to FcRn with a 2:1 ration (2 FcRn for eachantibody).

FIG. 6A to 6E shows different domain arrangements of bispecific proteinsproduced.

FIGS. 7A and 7B show respectively bispecific F1 and F2 antibodies havingNKp46 binding region based on NKp46-1, NKp46-2, NKp46-3 or NKp46-4 areable to direct resting NK cells to their CD19-positive Daudi tumortarget cells, while isotype control antibody did not lead to eliminationof the Daudi cells. Rituximab (RTX) served as positive control of ADCC,where the maximal response obtained with RTX (at 10 μg/ml in this assay)was 21.6% specific lysis.

FIG. 8A (top panel CD107; bottom panel CD69) shows bispecific antibodieshaving NKp46 and CD19 binding regions in an F2 format protein do notactivate resting NK cells in the absence of target cells, however fulllength anti-NKp46 antibodies as well as positive control alemtuzumab didactivate NK cells. FIG. 3B shows that in presence of Daudi target cells,bispecific anti-NKp46 x anti-CD19 antibodies (including each of theNKp46-1, NKp46-2, NKp46-3 or NKp46-4 binding domains) activated restingNK cells (top panel CD107, bottom panel CD69), while full-lengthanti-CD19 showed at best only very low activation of NK cells. Neitherfull-length anti-NKp46 antibodies or alemtuzumab showed substantialincrease in activation beyond what was observed in presence of NK cellsalone. FIG. 8C (top panel CD107, bottom panel CD69) shows that in thepresence of CD19-negative HUT78 cells, none of the bispecific anti-NKp46x anti-CD19 antibody (including each of the NKp46-1, NKp46-2, NKp46-3 orNKp46-4 variable regions) activated NK cells. However, the full-lengthanti-NKp46 antibodies and alemtuzumab caused detectable activation of NKcells at a similar level observed in presence of NK cells alone. Isotypecontrol antibody did not induce activation.

FIG. 9 shows that at low effector:target ratio of 1:1 each of thebispecific anti-NKp46 x anti-CD19 antibody activated NK cells in thepresence of Daudi cells, and that bispecific anti-NKp46 x anti-CD19 werefar more potent than the anti-CD19 antibody as a full-length human IgG1as ADCC inducing antibody. Top panel is CD107 and bottom panel showsCD69.

FIGS. 10A and 10B show that each NKp46 x CD19 bispecific protein (singlechain format F3, and multimeric formats F5 and F6) induced specificlysis of Daudi (FIG. 10A) or B221 (FIG. 10B) cells by human KHYG-1CD16-negative hNKp46-positive NK cell line, while rituximab and humanIgG1 isotype control (IC) antibodies did not.

FIG. 11 shows superimposed sensorgrams showing the binding of Macacafascicularis recombinant FcgRs (upper panels; CyCD64, CyCD32a, CYCD32b,CyCD16) and of human recombinant FcgRs (lower panels; HuCD64, HuCD32a,HuCD32b, HUCD16a) to the immobilized human IgG1 control (grey) andCD19/NKp46-1 bi-specific antibody with monomeric Fc domain (black).While full length wild type human IgG1 bound to all cynomolgus and humanFcγ receptors, the CD19/NKp46-1 monomeric-Fc bi-specific antibodies didnot bind to any of the receptors.

FIG. 12A shows results of purification by SEC of proteins format 6 (F6),compared with DART and BITE. BITE and DART showed a very low productionyield compared to F6 and have a very complex SEC profile. FIG. 12B showsSDS-PAGE after Coomassie staining in the expected SEC fractions (3 and 4for BITE and 4 and 5 for DART), whereas F6 format showed clear andsimple SEC and SDS-PAGE profiles with a major peak (fraction 3)containing the monomeric bispecific proteins.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used in the specification, “a” or “an” may mean one or more. As usedin the claim(s), when used in conjunction with the word “comprising”,the words “a” or “an” may mean one or more than one.

Where “comprising” is used, this can optionally be replaced by“consisting essentially of”, more optionally by “consisting of”.

As used herein, the term “antigen binding domain” refers to a domaincomprising a three-dimensional structure capable of immunospecificallybinding to an epitope. Thus, in one embodiment, said domain can comprisea hypervariable region, optionally a VH and/or VL domain of an antibodychain, optionally at least a VH domain. In another embodiment, thebinding domain may comprise at least one complementarity determiningregion (CDR) of an antibody chain. In another embodiment, the bindingdomain may comprise a polypeptide domain from a non-immunoglobulinscaffold.

The term “antibody” herein is used in the broadest sense andspecifically includes full-length monoclonal antibodies, polyclonalantibodies, multispecific antibodies (e.g., bispecific antibodies), andantibody fragments and derivatives, so long as they exhibit the desiredbiological activity. Various techniques relevant to the production ofantibodies are provided in, e.g., Harlow, et al., ANTIBODIES: ALABORATORY MANUAL, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., (1988). An “antibody fragment” comprises a portion of afull-length antibody, e.g. antigen-binding or variable regions thereof.Examples of antibody fragments include Fab, Fab′, F(ab)₂, F(ab′)₂,F(ab)₃, Fv (typically the VL and VH domains of a single arm of anantibody), single-chain Fv (scFv), dsFv, Fd fragments (typically the VHand CH1 domain), and dAb (typically a VH domain) fragments; VH, VL, VhH,and V-NAR domains; minibodies, diabodies, triabodies, tetrabodies, andkappa bodies (see, e.g., Ill et al., Protein Eng 1997; 10: 949-57);camel IgG; IgNAR; and multispecific antibody fragments formed fromantibody fragments, and one or more isolated CDRs or a functionalparatope, where isolated CDRs or antigen-binding residues orpolypeptides can be associated or linked together so as to form afunctional antibody fragment. Various types of antibody fragments havebeen described or reviewed in, e.g., Holliger and Hudson, Nat Biotechnol2005; 23, 1126-1136; WO2005040219, and published U.S. PatentApplications 20050238646 and 20020161201.

The term “antibody derivative”, as used herein, comprises a full-lengthantibody or a fragment of an antibody, e.g. comprising at leastantigen-binding or variable regions thereof, wherein one or more of theamino acids are chemically modified, e.g., by alkylation, PEGylation,acylation, ester formation or amide formation or the like. Thisincludes, but is not limited to, PEGylated antibodies,cysteine-PEGylated antibodies, and variants thereof.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody that are responsible for antigen binding.The hypervariable region generally comprises amino acid residues from a“complementarity-determining region” or “CDR” (e.g. residues 24-34 (L1),50-56 (L2) and 89-97 (L3) in the light-chain variable domain and 31-35(H1), 50-65 (H2) and 95-102 (H3) in the heavy-chain variable domain;Kabat et al. 1991) and/or those residues from a “hypervariable loop”(e.g. residues 26-32 (L1), 50-52 (L2) and 91-96 (L3) in the light-chainvariable domain and 26-32 (H1), 53-55 (H2) and 96-101 (H3) in theheavy-chain variable domain; Chothia and Lesk, J. Mol. Biol 1987;196:901-917). Typically, the numbering of amino acid residues in thisregion is performed by the method described in Kabat et al., supra.Phrases such as “Kabat position”, “variable domain residue numbering asin Kabat” and “according to Kabat” herein refer to this numbering systemfor heavy chain variable domains or light chain variable domains. Usingthe Kabat numbering system, the actual linear amino acid sequence of apeptide may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or CDR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of CDR H2and inserted residues (e.g. residues 82a, 82b, and 82c, etc. accordingto Kabat) after heavy chain FR residue 82. The Kabat numbering ofresidues may be determined for a given antibody by alignment at regionsof homology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

By “framework” or “FR” residues as used herein is meant the region of anantibody variable domain exclusive of those regions defined as CDRs.Each antibody variable domain framework can be further subdivided intothe contiguous regions separated by the CDRs (FR1, FR2, FR3 and FR4).

By “constant region” as defined herein is meant an antibody-derivedconstant region that is encoded by one of the light or heavy chainimmunoglobulin constant region genes. By “constant light chain” or“light chain constant region” as used herein is meant the region of anantibody encoded by the kappa (Ckappa) or lambda (Clambda) light chains.The constant light chain typically comprises a single domain, and asdefined herein refers to positions 108-214 of Ckappa, or Clambda,wherein numbering is according to the EU index (Kabat et al., 1991,Sequences of Proteins of Immunological Interest, 5th Ed., United StatesPublic Health Service, National Institutes of Health, Bethesda). By“constant heavy chain” or “heavy chain constant region” as used hereinis meant the region of an antibody encoded by the mu, delta, gamma,alpha, or epsilon genes to define the antibody's isotype as IgM, IgD,IgG, IgA, or IgE, respectively. For full length IgG antibodies, theconstant heavy chain, as defined herein, refers to the N-terminus of theCH1 domain to the C-terminus of the CH3 domain, thus comprisingpositions 118-447, wherein numbering is according to the EU index.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CH1, VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of apolypeptide, multispecific polypeptide or ABD, or any other embodimentsas outlined herein.

By “single-chain Fv” or “scFv” as used herein are meant antibodyfragments comprising the VH and VL domains of an antibody, wherein thesedomains are present in a single polypeptide chain. Generally, the Fvpolypeptide further comprises a polypeptide linker between the VH and VLdomains which enables the scFv to form the desired structure for antigenbinding. Methods for producing scFvs are well known in the art. For areview of methods for producing scFvs see Pluckthun in The Pharmacologyof Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds.Springer-Verlag, New York, pp. 269-315 (1994).

By “Fv” or “Fv fragment” or “Fv region” as used herein is meant apolypeptide that comprises the VL and VH domains of a single antibody.

By “Fc” or “Fc region”, as used herein is meant the polypeptidecomprising the constant region of an antibody excluding the firstconstant region immunoglobulin domain. Thus Fc refers to the last twoconstant region immunoglobulin domains of IgA, IgD, and IgG, and thelast three constant region immunoglobulin domains of IgE and IgM, andthe flexible hinge N-terminal to these domains. For IgA and IgM, Fc mayinclude the J chain. For IgG, Fc comprises immunoglobulin domains Cγ2(CH2) and Cγ3 (CH3) and the hinge between Cγ1 and Cγ2. Although theboundaries of the Fc region may vary, the human IgG heavy chain Fcregion is usually defined to comprise residues C226, P230 or A231 to itscarboxyl-terminus, wherein the numbering is according to the EU index.Fc may refer to this region in isolation, or this region in the contextof an Fc polypeptide, as described below. By “Fc polypeptide” or“Fc-derived polypeptide” as used herein is meant a polypeptide thatcomprises all or part of an Fc region. Fc polypeptides include but arenot limited to antibodies, Fc fusions and Fc fragments.

By “variable region” as used herein is meant the region of an antibodythat comprises one or more Ig domains substantially encoded by any ofthe VL (including Vkappa and Vlambda) and/or VH genes that make up thelight chain (including kappa and lambda) and heavy chain immunoglobulingenetic loci respectively. A light or heavy chain variable region (VLand VH) consists of a “framework” or “FR” region interrupted by threehypervariable regions referred to as “complementarity determiningregions” or “CDRs”. The extent of the framework region and CDRs havebeen precisely defined, for example as in Kabat (see “Sequences ofProteins of Immunological Interest,” E. Kabat et al., U.S. Department ofHealth and Human Services, (1983)), and as in Chothia. The frameworkregions of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs, which are primarily responsible for binding to an antigen.

The term “specifically binds to” means that an antibody or polypeptidecan bind preferably in a competitive binding assay to the bindingpartner, as assessed using either recombinant forms of the proteins,epitopes therein, or native proteins present on the surface of isolatedtarget cells. Competitive binding assays and other methods fordetermining specific binding are further described below and are wellknown in the art.

The term “affinity”, as used herein, means the strength of the bindingof an antibody or polypeptide to an epitope. The affinity of an antibodyis given by the dissociation constant K_(D), defined as[Ab]×[Ag]/[Ab−Ag], where [Ab−Ag] is the molar concentration of theantibody-antigen complex, [Ab] is the molar concentration of the unboundantibody and [Ag] is the molar concentration of the unbound antigen. Theaffinity constant K_(A) is defined by 1/K₀. Preferred methods fordetermining the affinity of mAbs can be found in Harlow, et al.,Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., 1988), Coligan et al., eds., Current Protocolsin Immunology, Greene Publishing Assoc. and Wiley Interscience, N.Y.,(1992, 1993), and Muller, Meth. Enzymol. 92:589-601 (1983), whichreferences are entirely incorporated herein by reference. One preferredand standard method well known in the art for determining the affinityof mAbs is the use of surface plasmon resonance (SPR) screening (such asby analysis with a BIAcore™ SPR analytical device).

By “amino acid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. An example ofamino acid modification herein is a substitution. By “amino acidmodification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence. By “amino acid substitution”or “substitution” herein is meant the replacement of an amino acid at agiven position in a protein sequence with another amino acid. Forexample, the substitution Y50W refers to a variant of a parentpolypeptide, in which the tyrosine at position 50 is replaced withtryptophan. A “variant” of a polypeptide refers to a polypeptide havingan amino acid sequence that is substantially identical to a referencepolypeptide, typically a native or “parent” polypeptide. The polypeptidevariant may possess one or more amino acid substitutions, deletions,and/or insertions at certain positions within the native amino acidsequence.

“Conservative” amino acid substitutions are those in which an amino acidresidue is replaced with an amino acid residue having a side chain withsimilar physicochemical properties. Families of amino acid residueshaving similar side chains are known in the art, and include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine), beta-branchedside chains (e.g., threonine, valine, isoleucine) and aromatic sidechains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

The term “identity” or “identical”, when used in a relationship betweenthe sequences of two or more polypeptides, refers to the degree ofsequence relatedness between polypeptides, as determined by the numberof matches between strings of two or more amino acid residues.“Identity” measures the percent of identical matches between the smallerof two or more sequences with gap alignments (if any) addressed by aparticular mathematical model or computer program (i.e., “algorithms”).Identity of related polypeptides can be readily calculated by knownmethods. Such methods include, but are not limited to, those describedin Computational Molecular Biology, Lesk, A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part 1, Griffin, A. M., and Griffin, H. G., eds., HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M.and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carilloet al., SIAM J. Applied Math. 48, 1073 (1988).

Preferred methods for determining identity are designed to give thelargest match between the sequences tested. Methods of determiningidentity are described in publicly available computer programs.Preferred computer program methods for determining identity between twosequences include the GCG program package, including GAP (Devereux etal., Nucl. Acid. Res. 12, 387 (1984); Genetics Computer Group,University of Wisconsin, Madison, Wis.), BLASTP, BLASTN, and FASTA(Altschul et al., J. Mol. Biol. 215, 403-410 (1990)). The BLASTX programis publicly available from the National Center for BiotechnologyInformation (NCBI) and other sources (BLAST Manual, Altschul et al.NCB/NLM/NIH Bethesda, Md. 20894; Altschul et al., supra). The well-knownSmith Waterman algorithm may also be used to determine identity.

An “isolated” molecule is a molecule that is the predominant species inthe composition wherein it is found with respect to the class ofmolecules to which it belongs (i.e., it makes up at least about 50% ofthe type of molecule in the composition and typically will make up atleast about 70%, at least about 80%, at least about 85%, at least about90%, at least about 95%, or more of the species of molecule, e.g.,peptide, in the composition). Commonly, a composition of a polypeptidewill exhibit 98%, 98%, or 99% homogeneity for polypeptides in thecontext of all present peptide species in the composition or at leastwith respect to substantially active peptide species in the context ofproposed use.

In the context herein, “treatment” or “treating” refers to preventing,alleviating, managing, curing or reducing one or more symptoms orclinically relevant manifestations of a disease or disorder, unlesscontradicted by context. For example, “treatment” of a patient in whomno symptoms or clinically relevant manifestations of a disease ordisorder have been identified is preventive or prophylactic therapy,whereas “treatment” of a patient in whom symptoms or clinically relevantmanifestations of a disease or disorder have been identified generallydoes not constitute preventive or prophylactic therapy.

As used herein, “NK cells” refers to a sub-population of lymphocytesthat is involved in non-conventional immunity. NK cells can beidentified by virtue of certain characteristics and biologicalproperties, such as the expression of specific surface antigensincluding CD56 and/or NKp46 for human NK cells, the absence of thealpha/beta or gamma/delta TCR complex on the cell surface, the abilityto bind to and kill cells that fail to express “self” MHC/HLA antigensby the activation of specific cytolytic machinery, the ability to killtumor cells or other diseased cells that express a ligand for NKactivating receptors, and the ability to release protein moleculescalled cytokines that stimulate or inhibit the immune response. Any ofthese characteristics and activities can be used to identify NK cells,using methods well known in the art. Any subpopulation of NK cells willalso be encompassed by the term NK cells. Within the context herein“active” NK cells designate biologically active NK cells, including NKcells having the capacity of lysing target cells or enhancing the immunefunction of other cells. NK cells can be obtained by various techniquesknown in the art, such as isolation from blood samples, cytapheresis,tissue or cell collections, etc. Useful protocols for assays involvingNK cells can be found in Natural Killer Cells Protocols (edited byCampbell K S and Colonna M). Human Press. pp. 219-238 (2000).

As used herein, “T cells” refers to a sub-population of lymphocytes thatmature in the thymus, and which display, among other molecules T cellreceptors on their surface. T cells can be identified by virtue ofcertain characteristics and biological properties, such as theexpression of specific surface antigens including the TCR, CD4 or CD8,the ability of certain T cells to kill tumor or infected cells, theability of certain T cells to activate other cells of the immune system,and the ability to release protein molecules called cytokines thatstimulate or inhibit the immune response. Any of these characteristicsand activities can be used to identify T cells, using methods well knownin the art. Within the context herein, “active” or “activated” T cellsdesignate biologically active T cells, more particularly T cells havingthe capacity of cytolysis or of stimulating an immune response by, e.g.,secreting cytokines. Active cells can be detected in any of a number ofwell-known methods, including functional assays and expression-basedassays such as the expression of cytokines such as TNF-alpha.

Producing Polypeptides

The antigen binding domains described herein can be readily derived avariety of immunoglobulin or non-immunoglobulin scaffolds, for exampleaffibodies based on the Z-domain of staphylococcal protein A, engineeredKunitz domains, monobodies or adnectins based on the 10th extracellulardomain of human fibronectin Ill, anticalins derived from lipocalins,DARPins (designed ankyrin repeat domains, multimerized LDLR-A module,avimers or cysteine-rich knottin peptides. See, e.g., Gebauer and Skerra(2009) Current Opinion in Chemical Biology 13:245-255, the disclosure ofwhich is incorporated herein by reference.

Variable domains are commonly derived from antibodies (immunoglobulinchains), for example in the form of associated VL and VH domains foundon two polypeptide chains, or single chain antigen binding domains suchas scFv, a V_(H) domain, a V_(L) domain, a dAb, a V-NAR domain or aV_(H)H domain. A variable domain can also be readily derived fromantibodies as a Fab.

Typically, antibodies are initially obtained by immunization of anon-human animal, e.g., a mouse, with an immunogen comprising apolypeptide, or a fragment or derivative thereof, typically animmunogenic fragment, for which it is desired to obtain antibodies (e.g.a human polypeptide). The step of immunizing a non-human mammal with anantigen may be carried out in any manner well known in the art forstimulating the production of antibodies in a mouse (see, for example,E. Harlow and D. Lane, Antibodies: A Laboratory Manual., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1988), the entiredisclosure of which is herein incorporated by reference). Otherprotocols may also be used as long as they result in the production of Bcells expressing an antibody directed to the antigen used inimmunization. Lymphocytes from a non-immunized non-human mammal may alsobe isolated, grown in vitro, and then exposed to the immunogen in cellculture. The lymphocytes are then harvested and the fusion stepdescribed below is carried out. For exemplary monoclonal antibodies, thenext step is the isolation of splenocytes from the immunized non-humanmammal and the subsequent fusion of those splenocytes with animmortalized cell in order to form an antibody-producing hybridoma. Thehybridoma colonies are then assayed for the production of antibodiesthat specifically bind to the polypeptide against which antibodies aredesired. The assay is typically a colorimetric ELISA-type assay,although any assay may be employed that can be adapted to the wells thatthe hybridomas are grown in. Other assays include radioimmunoassays orfluorescence activated cell sorting. The wells positive for the desiredantibody production are examined to determine if one or more distinctcolonies are present. If more than one colony is present, the cells maybe re-cloned and grown to ensure that only a single cell has given riseto the colony producing the desired antibody. After sufficient growth toproduce the desired monoclonal antibody, the growth media containingmonoclonal antibody (or the ascites fluid) is separated away from thecells and the monoclonal antibody present therein is purified.Purification is typically achieved by gel electrophoresis, dialysis,chromatography using protein A or protein G-Sepharose, or an anti-mouseIg linked to a solid support such as agarose or Sepharose beads (alldescribed, for example, in the Antibody Purification Handbook,Biosciences, publication No. 18-1037-46, Edition AC, the disclosure ofwhich is hereby incorporated by reference).

Human antibodies may also be produced by using, for immunization,transgenic animals that have been engineered to express a human antibodyrepertoire (Jakobovitz et Nature 362 (1993) 255), or by selection ofantibody repertoires using phage display methods. For example, aXenoMouse (Abgenix, Fremont, Calif.) can be used for immunization. AXenoMouse is a murine host that has had its immunoglobulin genesreplaced by functional human immunoglobulin genes. Thus, antibodiesproduced by this mouse or in hybridomas made from the B cells of thismouse, are already humanized. The XenoMouse is described in U.S. Pat.No. 6,162,963, which is herein incorporated in its entirety byreference.

Antibodies may also be produced by selection of combinatorial librariesof immunoglobulins, as disclosed for instance in (Ward et al. Nature,341 (1989) p. 544, the entire disclosure of which is herein incorporatedby reference). Phage display technology (McCafferty et al (1990) Nature348:552-553) can be used to produce antibodies from immunoglobulinvariable (V) domain gene repertoires from unimmunized donors. See, e.g.,Griffith et al (1993) EMBO J. 12:725-734; U.S. Pat. Nos. 5,565,332;5,573,905; 5,567,610; 5,229,275). When combinatorial libraries comprisevariable (V) domain gene repertoires of human origin, selection fromcombinatorial libraries will yield human antibodies.

Additionally, a wide range of antibodies are available in the scientificand patent literature, including DNA and/or amino acid sequences, orfrom commercial suppliers. Antibodies will typically be directed to apre-determined antigen. Examples of antibodies include antibodies thatrecognize an antigen expressed by a target cell that is to beeliminated, for example a proliferating cell or a cell contributing to apathology. Examples include antibodies that recognize tumor antigens,microbial (e.g. bacterial) antigens or viral antigens.

Variable domains and/or antigen binding domains can be selected based onthe desired cellular target, and may include for example cancerantigens, bacterial or viral antigens, etc. As used herein, the term“bacterial antigen” includes, but is not limited to, intact, attenuatedor killed bacteria, any structural or functional bacterial protein orcarbohydrate, or any peptide portion of a bacterial protein ofsufficient length (typically about 8 amino acids or longer) to beantigenic. Examples include gram-positive bacterial antigens andgram-negative bacterial antigens. In some embodiments the bacterialantigen is derived from a bacterium selected from the group consistingof Helicobacter species, in particular Helicobacter pyloris; Boreliaspecies, in particular Borelia burgdorferi; Legionella species, inparticular Legionella pneumophilia; Mycobacteria s species, inparticular M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M.gordonae; Staphylococcus species, in particular Staphylococcus aureus;Neisseria species, in particular N. gonorrhoeae, N. meningitidis;Listeria species, in particular Listeria monocytogenes; Streptococcusspecies, in particular S. pyogenes, S. agalactiae; S. faecalis; S.bovis, S. pneumonias; anaerobic Streptococcus species; pathogenicCampylobacter species; Enterococcus species; Haemophilus species, inparticular Haemophilus influenzue; Bacillus species, in particularBacillus anthracis; Corynebacterium species, in particularCorynebacterium diphtheriae; Erysipelothrix species, in particularErysipelothrix rhusiopathiae; Clostridium species, in particular C.perfringens, C. tetani; Enterobacter species, in particular Enterobacteraerogenes, Klebsiella species, in particular Klebsiella 1S pneumoniae,Pasteurella species, in particular Pasteurella multocida, Bacteroidesspecies; Fusobacterium species, in particular Fusobacterium nucleatum;Streptobacillus species, in particular Streptobacillus moniliformis;Treponema species, in particular Treponema pertenue; Leptospira;pathogenic Escherichia species; and Actinomyces species, in particularActinomyces israelli.

As used herein, the term “viral antigen” includes, but is not limitedto, intact, attenuated or killed whole virus, any structural orfunctional viral protein, or any peptide portion of a viral protein ofsufficient length (typically about 8 amino acids or longer) to beantigenic. Sources of a viral antigen include, but are not limited toviruses from the families: Retroviridae (e.g., human immunodeficiencyviruses, such as HIV-1 (also referred to as HTLV-III, LAV orHTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP;Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses,human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g.,strains that cause gastroenteritis); Togaviridae (e.g., equineencephalitis viruses, rubella viruses); Flaviviridae (e.g., dengueviruses, encephalitis viruses, yellow fever viruses); Coronaviridae(e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitisviruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenzaviruses); Bunyaviridae (e.g., Hantaan viruses, bunya viruses,phleboviruses and Nairo viruses); Arenaviridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviruses and rotaviruses);Bornaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae(parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses);Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus(HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpesvirus; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); andIridoviridae (e.g., African swine fever virus); and unclassified viruses(e.g., the agent of delta hepatitis (thought to be a defective satelliteof hepatitis B virus), Hepatitis C; Norwalk and related viruses, andastroviruses). Alternatively, a viral antigen may be producedrecombinantly.

As used herein, the terms “cancer antigen” and “tumor antigen” are usedinterchangeably and refer to antigens that are differentially expressedby cancer cells and can thereby be exploited in order to target cancercells. Cancer antigens are antigens which can potentially stimulateapparently tumor-specific immune responses. Some of these antigens areencoded, although not necessarily expressed, by normal cells. Theseantigens can be characterized as those which are normally silent (i.e.,not expressed) in normal cells, those that are expressed only at certainstages of differentiation and those that are temporally expressed suchas embryonic and fetal antigens. Other cancer antigens are encoded bymutant cellular genes, such as oncogenes (e.g., activated ras oncogene),suppressor genes (e.g., mutant p53), fusion proteins resulting frominternal deletions or chromosomal translocations. Still other cancerantigens can be encoded by viral genes such as those carried on RNA andDNA tumor viruses.

The cancer antigens are usually normal cell surface antigens which areeither overexpressed or expressed at abnormal times. Ideally the targetantigen is expressed only on proliferative cells (e.g., tumour cells),however this is rarely observed in practice. As a result, targetantigens are usually selected on the basis of differential expressionbetween proliferative and healthy tissue. Antibodies have been raised totarget specific tumour related antigens including: Receptor TyrosineKinase-like Orphan Receptor 1 (ROR1), Cripto, CD4, CD20, CD30, CD19,CD38, CD47, Glycoprotein NMB, CanAg, Her2 (ErbB2/Neu), CD22 (Siglec2),CD33 (Siglec3), CD79, CD138, CD171, PSCA, L1-CAM, PSMA (prostatespecific membrane antigen), BCMA, CD52, CD56, CD80, CD70, E-selectin,EphB2, Melanotransferin, Mud 6 and TMEFF2. Examples of cancer antigensalso include B7-H3, B7-H4, B7-H6, PD-L1, MAGE, MART-1/Melan-A, gp100,adenosine deaminase-binding protein (ADAbp), cyclophilin b, colorectalassociated antigen (CRC)-0017-1A/GA733, Killer-Ig Like Receptor 3DL2(KIR3DL2), protein tyrosine kinase 7 (PTK7), receptor protein tyrosinekinase 3 (TYRO-3), nectins (e.g. nectin-4), major histocompatibilitycomplex class I-related chain A and B polypeptides (MICA and MICB),proteins of the UL16-binding protein (ULBP) family, proteins of theretinoic acid early transcript-1 (RAET1) family, carcinoembryonicantigen (CEA) and its immunogenic epitopes CAP-1 and CAP-2, etv6, am11,prostate specific antigen (PSA), T-cell receptor/CD3-zeta chain,MAGE-family of tumor antigens, GAGE-family of tumor antigens,anti-Mullerian hormone Type II receptor, delta-like ligand 4 (DLL4),DR5, BAGE, RAGE, LAGE-1, NAG, GnT-V, MUM-1, CDK4, MUC family, VEGF, VEGFreceptors, Angiopoietin-2, PDGF, TGF-alpha, EGF, EGF receptor, a memberof the human EGF-like receptor family such as HER-2/neu, HER-3, HER-4 ora heterodimeric receptor comprised of at least one HER subunit, gastrinreleasing peptide receptor antigen, Muc-1, CA125, avβ3 integrins, a5β1integrins, αIIbβ3-integrins, PDGF beta receptor, SVE-cadherin, IL-8,hCG, IL-6, IL-6 receptor, IL-15, α-fetoprotein, E-cadherin, α-catenin,β-catenin and γ-catenin, p120ctn, PRAME, NY-ESO-1, cdc27, adenomatouspolyposis coli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15,gp75, GM2 and GD2 gangliosides, viral products such as humanpapillomavirus proteins, imp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, and c-erbB-2, although this is notintended to be exhaustive.

In one embodiment, an ABD, variable domain or pair of complementaryvariable domains binds to a cancer antigen, a viral antigen, a microbialantigen, or an antigen present on an infected cell (e.g. virallyinfected) or on a pro-inflammatory immune cell. In one embodiment, saidantigen is a polypeptide selectively expressed or overexpressed on atumor cell, and infected cell or a pro-inflammatory cell. In oneembodiment, said antigen is a polypeptide that when inhibited, decreasesthe proliferation and/or survival of a tumor cell, an infected cell or apro-inflammatory cell. For example, a first and/or second antibody orfragment can respectively bind anti-Her1 and anti-Her2. Anti-Her2 can befor example an antibody comprising the CDRs derived from Herceptin®(trastuzumab) or 2C4 (pertuzumab). Anti-Her2 and anti-Her1 (antibodiesD1-5 and C3-101) amino acid sequences are shown in WO2011/069104.

In one embodiment, an ABD, variable domain or pair of complementaryvariable domains inhibits (neutralizes) the function of a polypeptide towhich it specifically binds. In one embodiment, the first and/or secondABD each inhibits the function of a polypeptide to which theyspecifically binds. In one embodiment, the polypeptide is a polypeptideselectively expressed or overexpressed on a tumor cell. In oneembodiment, the polypeptide is a polypeptide selectively expressed oroverexpressed on an infected (e.g. virally or bacterially infected) cellor a pro-inflammatory cell. In one embodiment, the polypeptide is apolypeptide that when inhibited, decreases the proliferation and/orsurvival of a tumor cell, an infected cell or a pro-inflammatory cell.For example bispecific antibodies that bind ErbB2 and ErbB3 and blocksligand-induced receptor activation have been reported to be effective inErbB2-amplified tumors (MacDonagh et al. (2012) Mol. Cancer Ther.11:582).

In exemplary embodiments, one an ABD, variable domain or pair ofcomplementary variable domains will bind an antigen expressed by atarget cell that is to be eliminated (e.g., a tumor antigen, microbial(e.g. bacterial) antigen, viral antigen, or antigen expressed on animmune cell that is contributing to inflammatory or autoimmune disease,and the other ABD, variable domain or pair of complementary variabledomains will bind to an antigen expressed on an immune cell, for examplean immune effector cell, e.g. a cell surface receptor of an effectorcells such as a T or NK cell. Examples of antigens expressed on immunecells, optionally immune effector cells, include antigens expressed on amember of the human lymphoid cell lineage, e.g. a human T cell, a humanB cell or a human natural killer (NK) cell, a human monocyte, a humanneutrophilic granulocyte or a human dendritic cell. Advantageously, suchcells will have either a cytotoxic or an apoptotic effect on a targetcell that is to be eliminated (e.g., that expresses a tumor antigen,microbial antigen, viral antigen, or antigen expressed on an immune cellthat is contributing to inflammatory or autoimmune disease). Especiallyadvantageously, the human lymphoid cell is a cytotoxic T cell or NK cellwhich, when activated, exerts a cytotoxic effect on the target cell.According to this embodiment, then, the cytotoxic activity of the humaneffector cells is recruited. According to another embodiment, the humaneffector cell is a member of the human myeloid lineage.

Antigens expressed on an immune cell to which antibodies of fragmentsthat make up multispecific antibodies can bind also include NK and/or Tcell receptors, e.g. any molecule on the surface of NK cells or T cells,respectively, that can serve to direct the NK or T cell to the intendedtarget cell to be eliminated. Examples include, e.g., members of theimmunoglobulin superfamily, members of the killer-cellimmunoglobulin-like receptor (KIR) family, the leukocyteimmunoglobulin-like receptors (LILR) family, or the lectin family or theNK cell lectin-like receptor family. Activity can be measured forexample by bringing target cells and effector cells into contact inpresence of the multispecific polypeptide. Optionally the immune cellreceptor is an activating receptor, e.g. an activating NK cell or T cellreceptor. As used herein, the terms “activating NK cell receptor” and“activating T cell receptor” refers to any molecule on the surface of NKcells or T cells, respectively, that, when stimulated, causes ameasurable increase in any property or activity known in the art asassociated with NK cell or T cell activity, respectively, such ascytokine (for example IFN-γ or TNF-α) production, increases inintracellular free calcium levels, the ability to lyse target cells in aredirected killing assay as described, e.g. elsewhere in the presentspecification, or the ability to stimulate NK cell or T cellproliferation, respectively. The term “activating NK receptor” includesbut is not limited to DNAX accessory molecule-1 (DNAM-1), 2B4,activating forms of KIR proteins (for example KIR2DS receptors, KIR2DS2,KIR2DS4), NKG2D, NKp30, CD69, NKp80, NKp44, NKp46, IL-2R, IL-12R,IL-15R, IL-18R and IL-21R. In one embodiment, the activating NK cellreceptor is a receptor other than an FCγ receptor. In one embodiment,the activating NK cell receptor is a receptor other than NKp46.

Activation of cytotoxic T cells may occur via binding of the CD3 antigenas effector antigen on the surface of the cytotoxic T cell by amultispecific (e.g. bispecific) polypeptide of this embodiment. Thehuman CD3 antigen is present on both helper T cells and cytotoxic Tcells. Human CD3 denotes an antigen which is expressed on T cells aspart of the multimolecular T cell complex and which comprises threedifferent chains: CD3-epsilon, CD3-delta and CD3-gamma.

Other effector cell antigens bound by a multispecific polypeptide arethe human CD16 antigen, the human CD64, the human CD2 antigen, the humanCD28 antigen or the human CD25 antigen. In one embodiment, the effectorcell antigen is CD16; such a polypeptide, when having an Fc domain thatdoes not substantially bind inhibitory FcγR, will have CD16 agonistactivity without contribution of inhibition from inhibitory FcγR. Inother embodiments, the effector cell activating receptor is a receptorother than CD16.

The ABDs or variable domains which are incorporated into thepolypeptides can be tested for any desired activity prior to inclusionin a polypeptide. Once appropriate antigen binding domains havingdesired specificity and/or activity are identified, DNA encoding eachvariable domain can be placed, in suitable arrangements, in anappropriate expression vector(s), together with DNA encoding anyelements such as an enzymatic recognition tag, or CH2 and CH3 domainsand any other optional elements (e.g. DNA encoding a linker or hingeregion) for transfection into an appropriate host(s). The host is thenused for the recombinant production of the polypeptide chains that makeup the multispecific polypeptide.

An ABD or variable region derived from an antibody will generallycomprise at minimum a hypervariable region sufficient to confer bindingactivity when present in the multimeric polypeptide. It will beappreciated that an ABD or variable region may comprise other aminoacids or functional domains as may be desired, including but not limitedto linker elements (e.g. linker peptides, constant domain derivedsequences, hinges, or fragments thereof, each of which can be placedbetween a variable domain and a CH1, CL, CH2 or CH3 domain, or betweenother domains as needed).

In any embodiment, ABDs or variable regions can be obtained from ahumanized antibody in which residues from a complementary-determiningregion (CDR) of a human antibody are replaced by residues from a CDR ofthe original antibody (the parent or donor antibody, e.g. a murine orrat antibody) while maintaining the desired specificity, affinity, andcapacity of the original antibody. The CDRs of the parent antibody, someor all of which are encoded by nucleic acids originating in a non-humanorganism, are grafted in whole or in part into the beta-sheet frameworkof a human antibody variable region to create an antibody, thespecificity of which is determined by the engrafted CDRs. The creationof such antibodies is described in, e.g., WO 92/11018, Jones, 1986,Nature 321:522-525, Verhoeyen et al., 1988, Science 239:1534-1536. Anantigen binding domain can thus have non-human hypervariable regions orCDRs and human frameworks region sequences (optionally with backmutations).

Polypeptide chains will be arranged in one or more expression vectors soas to produce the polypeptides having the desired domains operablylinked to one another. A host cell chosen for expression of themultispecific polypeptide is an important contributor to the finalcomposition, including, without limitation, the variation in compositionof the oligosaccharide moieties decorating the protein in theimmunoglobulin CH2 domain. Thus, one aspect of the invention involvesthe selection of appropriate host cells for use and/or development of aproduction cell expressing the desired therapeutic protein such that themultispecific polypeptide retains at least partial FcRn binding but withdecreased binding to a Fcγ receptor compared, e.g., to a wild type fulllength human IgG1 antibody. The host cell may be of mammalian origin ormay be selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2,653, SP2/0, 293, HeLa, myeloma, lymphoma, yeast, insect or plant cells,or any derivative, immortalized or transformed cell thereof.Alternatively, the host cell may be selected from a species or organismincapable of glycosylating polypeptides, e.g. a prokaryotic cell ororganism, such as natural or engineered E. coli spp., Klebsiella spp.,or Pseudomonas spp.

The polypeptide can then be produced in an appropriate host cell or byany suitable synthetic process and brought into contact underappropriate conditions for the multimeric (e.g. dimeri or trimer)polypeptide to form.

Polypeptide Configurations

An isolated hetero-multimeric polypeptide that binds a first and secondantigen of interest in monovalent fashion can be prepared according todifferent configurations, in each case involving at least a central(first) polypeptide chain and a second polypeptide chain, and optionallya third polypeptide chain.

The first (central) polypeptide chain will provide one variable domainthat will, together with a complementary variable domain on a secondpolypeptide chain, form an antigen binding domain specific for one (e.g.a first) antigen of interest. The first (central) polypeptide chain willalso provide a second variable domain that will be paired with acomplementary variable domain to form an antigen binding domain specificfor another (e.g. a second) antigen of interest; the variable domainthat is complementary to the second variable domain can be placed on thecentral polypeptide (e.g. adjacent to the second variable domain in atandem variable domain construct such as an scFv), or can be placed onthe second polypeptide chain, or can be placed on a third polypeptidechain. The second (and third, if present) polypeptide chains willassociate with the central polypeptide chain by CH1-CKheterodimerization, forming interchain disulfide bonds betweenrespective hinge domains and between complementary CH1 and CK domains,with a single multimeric polypeptide being formed so long as CH/CK andVH/VK domains are chosen to give rise to a sole dimerizationconfiguration. In a trimer, or when polypeptides are constructed forpreparation of a trimer, there will generally be one polypeptide chainthat comprises a non-naturally occurring VH-CK or VL-CH1 domainarrangement.

The first (central) polypeptide chain comprises a first variable domain(V) fused to a CH1 of CL constant region (e.g. the V domain is fused atits C-terminus to the N-terminus of a CH1 or CK constant region), asecond variable domain, and an Fc domain (e.g. a full Fc domain or aportion thereof) interposed between the first and second variabledomains may have the Examples of domain arrangement for the firstpolypeptide include but are not limited to:

scFv - Fc domain - VH - CH1 scFv - Fc domain - VK - CK scFv - Fcdomain - VK - CH1 scFv - Fc domain - VH - CK (VH or VK) - Fc domain -VH - CH1 (VH or VK) - Fc domain - VK - CK (VH or VK) - Fc domain - VK -CH1 (VH or VK) - Fc domain - VH - CK (VH or VK) - CH1 - Fc domain - VH -CH1 (VH or VK) - CK - Fc domain - VK - CK (VH or VK) - CK - Fc domain -VK - CH1 (VH or VK) - CH1 - Fc domain - VH - CK (VH or VK) - CH1 - Fcdomain - VK - CH1 (VH or VK) - CK - Fc domain - VH - CK (VH or VK) -CK - Fc domain - VH - CH1 (VH or VK) - CH1 - Fc domain - VK - CK VH -CH1 - Fc domain - scFv VK - CK - Fc domain - scFv VH - CK - Fc domain -scFv VK - CH1 - Fc domain - scFv VH - CH1- Fc domain - (VH or VK) VK -CK - Fc domain - (VH or VK) VH - CK - Fc domain - (VH or VK) VK - CH1-Fc domain - (VH or VK) VH - CH1- Fc domain - CH1 - (VH or VK) VK - CK -Fc domain - CH1 - (VH or VK) VH - CK - Fc domain - CH1 - (VH or VK) VK -CH1- Fc domain - CH1 - (VH or VK) VH - CH1- Fc domain - CK - (VH or VK)VK - CK - Fc domain - CK - (VH or VK) VH - CK - Fc domain - CK - (VH orVK) VK - CH1- Fc domain - CK - (VH or VK)

A second polypeptide chain comprises a first variable domain (V) fused(e.g. at its C-terminus) to a CH1 or CL (e.g. CK) constant regionselected to be complementary to the CH1 or CL constant region of thefirst polypeptide chain such that the first and second polypeptides forma CH1-CL (e.g., CH1-CK) heterodimer. The second polypeptide chain mayfurther comprises an Fc domain (e.g. a full Fc domain or a portionthereof), e.g., fused to the C-terminus of the of the CH1 or CL domainor fused to the N-terminus of the variable domain. Examples of domainarrangement for the second polypeptide include but are not limited to:

(VH or VK) - (CH1) (VH or VK) - (CK) Fc domain - (VH or VK) - (CH1) Fcdomain - (VH or VK) - (CK) (VH or VK) - (CH1) - Fc domain (VH or VK) -(CK) - Fc domain (VH or VK) - Fc domain - (VH or VK) - (CH1) (VH orVK) - Fc domain - (VH or VK) - (CK)

A third polypeptide chain, when present, can have the domainarrangement: (VH or VK)-(CH1 or (CK).

Heterodimers

Examples of the domain arrangements (N- to C-terminal) of centralpolypeptide chains for use in such heterodimeric proteins include:

V_(a1)-(CH1 or CK)_(a)-Fc domain-V_(a2)-V_(b2);

V_(a2)-V_(b2)-Fc domain-V_(a1)-(CH1 or CK)_(a)

wherein V_(a1) is a light chain or heavy chain variable domain, andwherein one of V_(a2) and V_(b2) is a light chain variable domain andthe other is a heavy chain variable domain.

Further examples include:

V_(a1)-(CH1 or CK)_(a)-Fc domain-V_(b);

V_(b)-Fc domain-V_(a2)-(CH1 or CK)_(a)

wherein V_(b) binds antigen as a single variable domain (e.g. dAb, VhH).

The Fc domain of the central chain may be a full Fc domain (CH2-CH3) ora portion thereof sufficient to confer the desired functionality (e.g.FcRn binding). A second polypeptide chain will then be configured whichwill comprise an immunoglobulin variable domain and a CH1 or CK constantregion, e.g., a (CH1 or CK)_(b) unit, selected so as to permit CH1-CKheterodimerization with the central polypeptide chain; theimmunoglobulin variable domain will be selected so as to complement thevariable domain of the central chain that is adjacent to the CH1 or CKdomain, whereby the complementary variable domains form an antigenbinding domain for a first antigen of interest.

For example, a second polypeptide chain can comprise a domainarrangement:

V_(b1)-(CH1 or CK)_(b), or

V_(b1)-(CH1 or CK)_(b)-Fc domain

such that the (CH1 or CK)_(b) dimerizes with the (CH1 or CK)_(a) on thecentral chain, and the V_(b1) forms an antigen binding domain togetherwith V_(a1) of the central chain. If V_(a1) of the central chain is alight chain variable domain, V_(b1) will be a heavy chain variabledomain; and if V_(a1) of the central chain is a heavy chain variabledomain, V_(b1) will be a light chain variable domain.

The antigen binding domain for the second antigen of interest can thenbe formed from V_(a2) and V_(b2) which are configured as tandem variabledomains on the central chain forming the antigen binding domain for thesecond antigen of interest (e.g. a heavy chain variable domain (VH) anda light chain (kappa) variable domain (VK), for example forming an scFvunit). The antigen binding domain for the second antigen of interest canalso alternatively be formed from a single variable domain V₂ present onthe central chain.

The resulting heterodimer can for example have the configuration asfollows (see also Examples of such proteins shown as formats 2, 11 and12 shown in FIGS. 6A and 6C):

wherein one of V_(a1) of the first polypeptide chain and V_(b1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a2) and V_(b2)is a light chain variable domain and the other is a heavy chain variabledomain.

The resulting heterodimer can in another example have the configurationas follows (see also Examples of such proteins shown as format 10 shownin FIG. 6B):

wherein one of V_(a1) of the first polypeptide chain and V_(b1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a2) and V_(b2)is a light chain variable domain and the other is a heavy chain variabledomain.

The resulting heterodimer can in another example have the configurationas follows (see also Examples of such proteins shown as formats 13 and14 shown in FIGS. 6D and 6E):

wherein one of V_(a1) of the first polypeptide chain and V_(b1) of thesecond polypeptide chain is a light chain variable domain and the otheris a heavy chain variable domain, and wherein one of V_(a2) and V_(b2)is a light chain variable domain and the other is a heavy chain variabledomain.

In one embodiment, the heterodimeric bispecific Fc-derived polypeptidecomprises a domain arrangement of one of the following, optionallywherein one or both hinge domains are replaced by a peptide linker,optionally wherein the Fc domain is fused via a peptide linker to anscFv that binds a polypeptide expressed by an immune effector cell (e.g.T cell, NK cell, etc.):

Examples of domain arrangement for the dimeric polypeptide formedinclude but are not limited to those in the table below:

Heterotrimers

Heterotrimeric proteins can for example be formed by using a central(first) polypeptide chain comprising a first variable domain (V) fusedto a first CH1 or CK constant region, a second variable domain (V) fusedto a second CH1 or CK constant region, and an Fc domain or portionthereof interposed between the first and second variable domains (i.e.the Fc domain is interposed between the first and second (V-(CH1/CK))units. For example, a central polypeptide chain for use in aheterotrimeric protein can have the domain arrangements (N- toC-terminal) as follows:

V_(a1)-(CH1 or CK)_(a)-Fc domain-V_(a2)-(CH1 or CK)_(b).

A second polypeptide chain can then comprise a domain arrangement (N- toC-terminal):

V_(b1)-(CH1 or CK)_(c),

or

V_(b1)-(CH1 or CK)_(c)-Fc domain

such that the (CH1 or CK)_(c) dimerizes with the (CH1 or CK)_(a) on thecentral chain, and the V_(a1) and V_(b1) form an antigen binding domain.

A third polypeptide chain can then comprise a domain arrangement (N- toC-terminal):

V_(b2)-(CH1 or CK)_(d),

such that the (CH1 or CK)_(d) dimerizes with the (CH1 or CK)_(b) unit onthe central chain, and the V_(at) and V_(b2) form an antigen bindingdomain.

An example of a configuration of a resulting heterotrimer with a dimericFc domain (also shown as formats 5, 6, 7 and 16 in FIGS. 6D and 6E) hasa domain arrangement:

An example of a configuration of a resulting heterotrimer with amonomeric Fc domain (also shown as formats 8, 9 and 17 in FIGS. 6B and6C) has a domain arrangement:

Thus, in a configuration of a trimer polypeptide, the first polypeptidecan have two variable domains that each form an antigen binding domainwith a variable domain on a separate polypeptide chain (i.e. thevariable domain of the second and third chains), the second polypeptidechain has one variable domain, and the third polypeptide has onevariable domain.

A trimeric polypeptide may comprise:

(a) a first polypeptide chain comprising a first variable domain (V)fused to a first CH1 of CK constant region, a second variable domain (V)fused to a second CH1 of CK constant region, and an Fc domain or portionthereof interposed between the first and second variable domains;

(b) a second polypeptide chain comprising a variable domain fused at itsC-terminus to a CH1 or CK constant region selected to be complementaryto the first CH1 or CK constant region of the first polypeptide chainsuch that the first and second polypeptides form a CH1-CK heterodimer,and optionally an Fc domain; and

(c) a third polypeptide chain comprising a variable domain fused (e.g.at its C-terminus) to a CH1 or CK constant region, wherein the variabledomain and the constant region are selected to be complementary to thesecond variable domain and second CH1 or CK constant region of the firstpolypeptide chain such that the first and third polypeptides form aCH1-CK heterodimer bound by disulfide bond(s) formed between the CH1 orCK constant region of the third polypeptide and the second CH1 or CKconstant region of the first polypeptide, but not between the CH1 or CKconstant region of the third polypeptide and the first CH1 or CKconstant region of the first polypeptide

wherein the first, second and third polypeptides form a CH1-CKheterotrimer, and wherein the first variable domain of the firstpolypeptide chain and the variable domain of the second polypeptidechain form an antigen binding domain specific for a first antigen ofinterest, and the second variable domain of the first polypeptide chainand the variable domain on the third polypeptide chain form an antigenbinding domain specific for a second antigen of interest.

Examples of domain arrangement for the trimeric bispecific polypeptideformed from include but are not limited to:

A hinge region will typically be present on a polypeptide chain betweena CH1 domain and a CH2 domain, and/or can be present between a CK domainand a CH2 domain. A hinge region can optionally be replaced for exampleby a suitable linker peptide.

The proteins domains described in the present disclosure can optionallybe specified as being from N- to C-terminal. Protein arrangements of thedisclosure for purposes of illustration are shown from N-terminus (onthe left) to C-terminus. Domains can be referred to as fused to oneanother (e.g. a domain can be said to be fused to the C-terminus of thedomain on its left, and/or a domain can be said to be fused to theN-terminus of the domain on its right).

The proteins domains described in the present disclosure can be fused toone another directly or via intervening amino acid sequences. Forexample, a CH1 or CK domain will be fused to an Fc domain (or CH2 or CH3domain thereof) via a linker peptide, optionally a hinge region or afragment thereof. In another example, a VH or VK domain will be fused toa CH3 domain via a linker peptide. VH and VL domains linked to anotherin tandem will be fused via a linker peptide (e.g. as an scFv). VH andVL domains linked to an Fc domain will be fused via a linker peptide.Two polypeptide chains will be bound to one another (indicated by “|”),preferably by interchain disulfide bonds formed between cysteineresidues within complementary CH1 and CK domains.

It will be appreciated that in any embodiment herein, a VK domain can bereplaced by a VA variable domain.

In any of the domain arrangements, the Fc domain may comprise a CH2-CH3unit (a full length CH2 and CH3 domain or a fragment thereof). Inheterodimers or heterotrimers comprising two chains with Fc domains (adimeric Fc domain), the CH3 domain will be capable of CH3-CH3dimerization (e.g. a wild-type CH3 domain). In heterodimers orheterotrimers comprising only one chain with an Fc domain (monomeric Fcdomain), the Fc domain will be incapable of CH3-CH3 dimerization; forexample the CH3 domain(s) will have amino acid modification(s) in theCH3 dimer interface or the Fc domain will comprise a tandem CH3 domainincapable of CH3-CH3 dimerization. In one embodiment of any aspectherein, a first CH3 domain is connected to a second CH3 domain by alinker. The tandem CH3 domain may have the domain arrangement, fromN-terminus to C-terminus, as follows:

—CH3-linker-CH3-.

The linker in the tandem CH3 domain will be a flexible linker (e.g.peptide linker). In one embodiment the linker permits the CH3 domains toassociate with one another by non-covalent interactions. In oneembodiment, the linker is a peptide linker having 10-50 amino acidresidues. In one embodiment, the linker has the formula (G₄S)_(x).Optionally, x is 2, 3, 4, 5 or 6. In any of the embodiments, each CH3domain is independently a full-length and/or native CH3 domain, or afragment or modified CH3 domain which retains a functional CH3dimerization interface.

An exemplary tandem CH3 with a flexible peptide linker (underlined) isshown below. An exemplary tandem CH3 domain can thus comprise an aminoacid sequence of SEQ ID NO: 2, or a sequence at least 70%, 80%, 90%, 95%or 98% identical thereto:

(SEQ ID NO: 2) G Q P R E P Q V Y T L P p S R E E M T KN Q V S L T C L V K G F Y P S D I A V EW E S N G Q P E N N Y K T T P P V L D SD G S F F L Y S K L T V D K S R w Q Q GN V F S C S V M H E A L H N H Y T Q K S L S L S P G G G G G S G G G G s G G G G S  G Q P R E P Q V Y T L P P S R E E M TK N Q V S L T C L V K G F Y P S D I A VE W E S N G Q P E N N Y K T T P P V L DS D G S F F L Y S K L T V D K S R w Q QG N V F S C S V M H E A L H N H Y T Q K S L S L S P G

Tandem CH3 domains disclosed herein and CH3 domains with amino acidmodification to prevent CH3-CH3 dimerization will retain partial FcRnbinding (compared, e.g., to a wild type full length human IgG1antibody). The examples of monomeric CH2-CH3 domains provided hereinretain partial FcRn binding but have decreased human Fcγ receptorbinding. Optionally the multimeric polypeptide is capable of binding tohuman FcRn with intermediate affinity, e.g. retains binding to FcRn buthas decreased binding to a human FcRn receptor compared to a full-lengthwild type human IgG1 antibody. The Fc moiety may further comprise one ormore amino acid modifications, e.g. in the CH2 domain, that decreasesfurther (e.g. abolishes) binding to one or more Fcγ receptors.

The multimeric polypeptides with monomeric Fc domains can advantageouslycomprise a CH2 domain and a CH3 domain, wherein said CH3 domaincomprises a modified CH3 dimer interface (e.g. a mutations in the CH3dimer interface) to prevent dimerization with another Fc-derivedpolypeptide. In one embodiment a CH2-CH3 portion comprising a CH3 domainmodified to prevent homodimer formation comprises an amino acid sequenceof SEQ ID NO: 1, or a sequence at least 90, 95% or 98% identicalthereto: APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 1), optionally comprisinga substitution at 1, 2, 3, 4, 5, 6 of residues 121, 136, 165, 175, 177or 179 of SEQ ID NO: 1.

In one embodiment of any of the polypeptides or methods herein, the CH3domain comprises an amino acid substitution at 1, 2, 3, 4, 5, 6 or 7 ofthe positions L351, T366, L368, P395, F405, T407 (or Y407) and/or K409(EU numbering as in Kabat).

In one embodiment, a peptide linker used to link a variable domain to aCH2 or CH3 comprises a fragment of a CH1 domain and/or hinge region. Forexample, a N-terminal amino acid sequence of CH1 can be fused to avariable domain in order to mimic as closely as possible the naturalstructure of an antibody. In one embodiment, the linker comprises aN-terminal CH1 amino acid sequence of between 2-4 residues, between 2-4residues, between 2-6 residues, between 2-8 residues, between 2-10residues, between 2-12 residues, between 2-14 residues, between 2-16residues, between 2-18 residues, between 2-20 residues, between 2-22residues, between 2-24 residues, between 2-26 residues, between 2-28residues, or between 2-30 residues. In one embodiment linker comprisesor consists of the amino acid sequence RTVA.

When two variable regions form a scFv they are linked together by alinker of sufficient length to enable the ABD to fold in such a way asto permit binding to the antigen for which the ABD is intended to bind.Examples of linkers include, for example, linkers comprising glycine andserine residues, e.g., the amino acid sequence GEGTSTGS(G₂S)₂GGAD. Inanother specific embodiment, the VH domain and VL domains of an svFv arelinked together by the amino acid sequence (G₄S)₃.

Any of the peptide linkers may comprise a length of at least 5 residues,at least 10 residues, at least 15 residues, at least 20 residues, atleast 25 residues, at least 30 residues or more. In other embodiments,the linkers comprises a length of between 2-4 residues, between 2-4residues, between 2-6 residues, between 2-8 residues, between 2-10residues, between 2-12 residues, between 2-14 residues, between 2-16residues, between 2-18 residues, between 2-20 residues, between 2-22residues, between 2-24 residues, between 2-26 residues, between 2-28residues, or between 2-30 residues.

Constant region domains can be derived from any suitable antibody. Ofparticular interest are the heavy chain domains, including, the constantheavy (CH) domains and the hinge domains. In the context of IgGantibodies, the IgG isotypes each have three CH regions. Accordingly,“CH” domains in the context of IgG are as follows: “CH1” refers topositions 118-220 according to the EU index as in Kabat. “CH2” refers topositions 237-340 according to the EU index as in Kabat, and “CH3”refers to positions 341-447 according to the EU index as in Kabat. By“hinge” or “hinge region” or “antibody hinge region” is meant theflexible polypeptide comprising the amino acids between the first andsecond constant domains of an antibody. Structurally, the IgG CH1 domainends at EU position 220, and the IgG CH2 domain begins at residue EUposition 237. Thus for IgG the hinge is herein defined to includepositions 221 (D221 in IgG1) to 236 (G236 in IgG1), wherein thenumbering is according to the EU index as in Kabat. References to aminoacid residue within constant region domains found within thepolypeptides shall be, unless otherwise indicated or as otherwisedictated by context, with reference to Kabat, in the context of an IgGantibody. CH3 domains that can serve in the present antibodies can bederived from any suitable antibody. Such CH3 domains can serve as thebasis for a modified CH3 domain. Optionally the CH3 domain is of humanorigin.

In certain embodiments herein where a multimeric polypeptide comprises amonomeric Fc domain, the CH3 domain will comprise one or more amino acidmodifications (e.g. amino acid substitutions) to disrupt the CH3dimerization interface. Optionally the CH3 domain modifications willprevent protein aggregation caused by the exposure of hydrophobicresidues when the CH2-CH3 domains are in monomeric form. Optionally, theCH3 domain modifications will additionally not interfere with theability of the Fc-derived polypeptide to bind to neonatal Fc receptor(FcRn), e.g. human FcRn.

CH3 domains that can be used to prevent CH3-CH3 dimerization have beendescribed in various publications. See, e.g. US 2006/0074225,WO2006/031994, WO2011/063348 and Ying et al. (2012) J. Biol. Chem.287(23):19399-19407, the disclosures of each of which are incorporatedherein by reference. In order to discourage the homodimer formation, oneor more residues that make up the CH3-CH3 interface are replaced with acharged amino acid such that the interaction becomes electrostaticallyunfavorable. For example, WO2011/063348 provides that a positive-chargedamino acid in the interface, such as lysine, arginine, or histidine, isreplaced with a different (e.g. negative-charged amino acid, such asaspartic acid or glutamic acid), and/or a negative-charged amino acid inthe interface is replaced with a different (e.g. positive charged) aminoacid. Using human IgG as an example, charged residues within theinterface that may be changed to the opposing charge include R355, D356,E357, K370, K392, D399, K409, and K439. In certain embodiments, two ormore charged residues within the interface are changed to an oppositecharge. Exemplary molecules include those comprising K392D and K409Dmutations and those comprising D399K and D356K mutations. In order tomaintain stability of the Fc domain in monomeric form, one or more largehydrophobic residues that make up the CH3-CH3 interface are replacedwith a small polar amino acid. Using human IgG as an example, largehydrophobic residues of the CH3-CH3 interface include Y349, L351, L368,L398, V397, F405, and Y407. Small polar amino acid residues includeasparagine, cysteine, glutamine, serine, and threonine. Thus in oneembodiment, a CH3 domain will comprise an amino acid modification (e.g.substitution) at 1, 2, 3, 4, 5, 6, 7 or 8 of the positions R355, D356,E357, K370, K392, D399, K409, and K439. In WO2011/063348, two of thepositively charged Lys residues that are closely located at the CH3domain interface were mutated to Asp. Threonine scanning mutagenesis wasthen carried out on the structurally conserved large hydrophobicresidues in the background of these two Lys to Asp mutations. Fcmolecules comprising K392D and K409D mutations along with the varioussubstitutions with threonine were analyzed for monomer formation.Exemplary monomeric Fc domains include those having K392D, K409D andY349T substitutions and those having K392D, K409D and F405Tsubstitutions.

In Ying et al. (2012) J. Biol. Chem. 287(23):19399-19407, amino acidsubstitutions were made within the CH3 domain at residues L351, T366,L368, P395, F405, T407 and K409. Combinations of different mutationsresulted in the disruption of the CH3 dimerization interface, withoutcausing protein aggregation. Thus in one embodiment, a CH3 domain willcomprise an amino acid modification (e.g. substitution) at 1, 2, 3, 4,5, 6 or 7 of the positions L351, T366, L368, P395, F405, T407 and/orK409. In one embodiment, a CH3 domain will comprise amino acidmodifications L351Y, T366Y, L368A, P395R, F405R, T407M and K409A. In oneembodiment, a CH3 domain will comprise amino acid modifications L351S,T366R, L368H, P395K, F405E, T407K and K409A. In one embodiment, a CH3domain will comprise amino acid modifications L351K, T366S, P395V,F405R, T407A and K409Y.

CH2 domains can be readily obtained from any suitable antibody.Optionally the CH2 domain is of human origin. A CH2 may or may not belinked (e.g. at its N-terminus) to a hinge of linker amino acidsequence. In one embodiment, a CH2 domain is a naturally occurring humanCH2 domain of IgG1, 2, 4 or 4 subtype. In one embodiment, a CH2 domainis a fragment of a CH2 domain (e.g. at least 10, 20, 30, 40 or 50 aminoacids).

In one embodiment, a CH2 domain, when present in a polypeptide describedherein, will retain binding to a neonatal Fc receptor (FcRn),particularly human FcRn.

In one embodiment, a CH2 domain, when present in a polypeptide describedherein, and the polypeptides described herein, will confer decreased orlack of binding to a Fcγ receptor, notably FcγRIIIA (CD16). Polypeptidesthat comprise a CH2 domain that are not bound by CD16 will not becapable of activating or mediating ADCC by cells (e.g. NK cells, Tcells) that do not express the effector cell antigen of interest (e.g.NKp46, CD3, etc.).

In one embodiment, the polypeptides described herein and their Fcdomain(s) and/or a CH2 domain thereof, will have decreased or willsubstantially lack antibody dependent cytotoxicity (ADCC), complementdependent cytotoxicity (CDC), antibody dependent cellular phagocytosis(ADCP), FcR-mediated cellular activation (e.g. cytokine release throughFcR cross-linking), and/or FcR-mediated platelet activation/depletion,as mediated by immune cells (e.g. effector cells) that do not expressthe antigen of interest.

In one embodiment, a CH2 domain in a polypeptide will have substantialloss of binding to activating Fcγ receptors, e.g., FcγRIIIA (CD16),FcγRIIA (CD32A) or CD64, or to an inhibitory Fc receptor, e.g., FcγRIIB(CD32B). In one embodiment, a CH2 domain in a polypeptide willfurthermore have substantial loss of binding to the first component ofcomplement (C1q).

For example, substitutions into human IgG1 of IgG2 residues at positions233-236 and IgG4 residues at positions 327, 330 and 331 were shown togreatly reduce binding to Fcγ receptors and thus ADCC and CDC.Furthermore, Idusogie et al. (2000) J Immunol. 164(8):4178-84demonstrated that alanine substitution at different positions, includingK322, significantly reduced complement activation.

In one embodiment, a CH2 domain that retains binding to a Fcγ receptorbut has reduction of binding to Fcγ receptors will lack or have modifiedN-linked glycosylation, e.g. at residue N297 (Kabat EU). For example thepolypeptide is expressed in a cell line which naturally has a highenzyme activity for adding fucosyl to the N-acetylglucosamine that bindsto the Fc region of the polypeptides, or which does not yieldglycosylation at N297 (e.g. bacterial host cells). In anotherembodiment, a polypeptide may have one or more substitution that resultin lack of the canonical Asn-X-Ser/Thr N-linked glycosylation motif atresidues 297-299, which can also thus also result in reduction ofbinding to Fcγ receptors. Thus, a CH2 domain may have a substitution atN297 and/or at neighboring residues (e.g. 298, 299).

In one embodiment, an Fc domain or a CH2 domain therefrom is derivedfrom an IgG2 Fc mutant exhibiting diminished FcγR binding capacity buthaving conserved FcRn binding. In one aspect, the IgG2 Fc mutant or thederived multispecific polypeptide, Fc domain or CH2 domain comprises themutations V234A, G237A, P238S according to the EU numbering system. Inanother aspect, the IgG2 Fc mutant or the derived multispecificpolypeptide or Fc domain comprises mutations V234A, G237A, H268Q orH268A, V309L, A330S, P331S according to the EU numbering system. In aparticular aspect, the IgG2 Fc mutant or the derived multispecificpolypeptide or Fc domain comprises mutations V234A, G237A, P238S, H268A,V309L, A330S, P331S, and, optionally, P233S according to the EUnumbering system. Optionally, a CH2 domain with loss of binding to Fcγreceptors may comprises residues 233, 234, 235, 237, and 238 (EUnumbering system) that comprise a sequence selected from PAAAP, PAAAS,and SAAAS, optionally an Fc domain having such mutations can furthercomprise mutations H268A or H268Q, V309L, A330S and P331S (seeWO2011/066501, the disclosure of which is incorporated herein byreference).

In one embodiment, a CH2 domain that loses binding to a Fcγ receptorwill comprise at least one amino acid modification (for example,possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acid modifications)in the CH2 domain of the Fc region, optionally further in combinationwith one or more amino acid modification in other domains (e.g. in ahinge domain or a CH3 domain). Any combination of Fc modifications canbe made, for example any combination of different modificationsdisclosed in Armour KL. et al., (1999) Eur J Immunol. 29(8):2613-24;Presta, L. G. et al. (2002) Biochem. Soc. Trans. 30(4):487-490; Shields,R. L. et al. (2002) J. Biol. Chem. 26; 277(30):26733-26740 and Shields,R. L. et al. (2001) J. Biol. Chem. 276(9):6591-6604). In one embodiment,a polypeptide of the invention that has decreased binding to a human Fcγreceptor will comprise at least one amino acid modification (forexample, possessing 1, 2, 3, 4, 5, 6, 7, 8, 9, or more amino acidmodifications) relative to a wild-type CH2 domain within amino acidresidues 237-340 (EU numbering), such that the polypeptide comprisingsuch CH2 domain has decreased affinity for a human Fcγ receptor ofinterest relative to an equivalent polypeptide comprising a wild-typeCH2 domain, optionally wherein the variant CH2 domain comprises asubstitution at any one or more of positions 233, 234, 235, 236, 237,238, 268, 297, 238, 299, 309, 327, 330, 331 (EU numbering).

In one aspect, provided is an isolated multispecific F2 to F17heterodimeric protein comprising a first polypeptide chain comprising afirst amino acid sequence which is at least 50%, 60%, 70%, 80%, 85%,90%, 95% or 98% identical to the sequence of a first polypeptide chainof a F2 to F17 polypeptides disclosed herein; and a second amino acidsequence which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98%identical to the sequence of a second polypeptide chain of therespective F2 to F17 polypeptide disclosed herein. Optionally any or allof the variable regions or CDRs of the first and second chains aresubstituted with different variable regions, optionally where variableregions are excluded from the sequences that are considered forcomputing identity.

In one aspect, provided is an isolated multispecific heterotrimericprotein comprising a first polypeptide chain comprising a first aminoacid sequence which is at least 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98%identical to the sequence of a first polypeptide chain of the F2 to F17polypeptides disclosed herein; a second amino acid sequence which is atleast 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98% identical to the sequenceof a second polypeptide chain of the respective F2 to F17 polypeptidedisclosed herein; and a third amino acid sequence which is at least 50%,60%, 70%, 80%, 85%, 90%, 95% or 98% identical to the sequence of a thirdpolypeptide chain of the respective F2 to F17 polypeptide disclosedherein. Optionally any or all of the variable regions or CDRs of thefirst and second chains are substituted with different variable regions,optionally where variable regions are excluded from the sequences thatare considered for computing identity.

Uses of Compounds

In one aspect, provided are the use of any of the compounds definedherein for the manufacture of a pharmaceutical preparation for thetreatment or diagnosis of a mammal being in need thereof. Provided alsoare the use any of the compounds defined above as a medicament or anactive component or active substance in a medicament. In a furtheraspect provided is a method for preparing a pharmaceutical compositioncontaining a compound as defined above, to provide a solid or a liquidformulation for administration orally, topically, or by injection. Sucha method or process at least comprises the step of mixing the compoundwith a pharmaceutically acceptable carrier.

In one aspect, provided is a method to treat, prevent or more generallyaffect a predefined condition by exerting a certain effect, or detect acertain condition using a compound herein, or a (pharmaceutical)composition comprising a compound disclosed herein.

The polypeptides described herein can be used to prevent or treatdisorders that can be treated with antibodies, such as cancers, solidand non-solid tumors, hematological malignancies, infections such asviral infections, and inflammatory or autoimmune disorders. In oneembodiment, the an antigen of interest expressed on the surface of amalignant cell of a type cancer selected from the group consisting of:carcinoma, including that of the bladder, head and neck, breast, colon,kidney, liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroidand skin, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma, hematopoietic tumors of lymphoidlineage, for example T-cell and B-cell tumors, including but not limitedto T-cell disorders such as T-prolymphocytic leukemia (T-PLL), includingof the small cell and cerebriform cell type; large granular lymphocyteleukemia (LGL) preferably of the T-cell type; Sezary syndrome (SS);Adult T-cell leukemia lymphoma (ATLL); a/d T-NHL hepatosplenic lymphoma;peripheral/post-thymic T cell lymphoma (pleomorphic and immunoblasticsubtypes); angio immunoblastic T-cell lymphoma; angiocentric (nasal)T-cell lymphoma; anaplastic (Ki 1+) large cell lymphoma; intestinalT-cell lymphoma; T-lymphoblastic; and lymphoma/leukaemia (T-Lbly/T-ALL).

In one embodiment, polypeptides described herein can be used to preventor treat a cancer selected from the group consisting of: carcinoma,including that of the bladder, head and neck, breast, colon, kidney,liver, lung, ovary, prostate, pancreas, stomach, cervix, thyroid andskin, including squamous cell carcinoma; hematopoietic tumors oflymphoid lineage, including leukemia, acute lymphocytic leukemia, acutelymphoblastic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkinslymphoma, non-Hodgkins lymphoma, hairy cell lymphoma and Burkettslymphoma; hematopoietic tumors of myeloid lineage, including acute andchronic myelogenous leukemias and promyelocytic leukemia; tumors ofmesenchymal origin, including fibrosarcoma and rhabdomyoscarcoma; othertumors, including neuroblastoma and glioma; tumors of the central andperipheral nervous system, including astrocytoma, neuroblastoma, glioma,and schwannomas; tumors of mesenchymal origin, including fibrosarcoma,rhabdomyoscaroma, and osteosarcoma; and other tumors, includingmelanoma, xeroderma pigmentosum, keratoacanthoma, seminoma, thyroidfollicular cancer and teratocarcinoma. Other exemplary disorders thatcan be treated according to the invention include hematopoietic tumorsof lymphoid lineage, for example T-cell and B-cell tumors, including butnot limited to T-cell disorders such as T-prolymphocytic leukemia(T-PLL), including of the small cell and cerebriform cell type; largegranular lymphocyte leukemia (LGL) preferably of the T-cell type; Sezarysyndrome (SS); Adult T-cell leukemia lymphoma (ATLL); a/d T-NHLhepatosplenic lymphoma; peripheral/post-thymic T cell lymphoma(pleomorphic and immunoblastic subtypes); angio immunoblastic T-celllymphoma; angiocentric (nasal) T-cell lymphoma; anaplastic (Ki 1+) largecell lymphoma; intestinal T-cell lymphoma; T-lymphoblastic; andlymphoma/leukaemia (T-Lbly/T-ALL).

In one aspect, the methods of treatment comprise administering to anindividual a multispecific polypeptide in a therapeutically effectiveamount. A therapeutically effective amount may be any amount that has atherapeutic effect in a patient having a disease or disorder (orpromotes, enhances, and/or induces such an effect in at least asubstantial proportion of patients with the disease or disorder andsubstantially similar characteristics as the patient).

The multispecific polypeptides can be included in kits. The kits mayoptionally further contain any number of polypeptides and/or othercompounds, e.g., 1, 2, 3, 4, or any other number of multispecificpolypeptide and/or other compounds. It will be appreciated that thisdescription of the contents of the kits is not limiting in any way. Forexample, the kit may contain other types of therapeutic compounds.Optionally, the kits also include instructions for using thepolypeptides, e.g., detailing the herein-described methods.

Also provided are pharmaceutical compositions comprising the compoundsas defined above. A compound may be administered in purified formtogether with a pharmaceutical carrier as a pharmaceutical composition.The form depends on the intended mode of administration and therapeuticor diagnostic application. The pharmaceutical carrier can be anycompatible, nontoxic substance suitable to deliver the compounds to thepatient. Pharmaceutically acceptable carriers are well known in the artand include, for example, aqueous solutions such as (sterile) water orphysiologically buffered saline or other solvents or vehicles such asglycols, glycerol, oils such as olive oil or injectable organic esters,alcohol, fats, waxes, and inert solids A pharmaceutically acceptablecarrier may further contain physiologically acceptable compounds thatact for example to stabilize or to increase the absorption of thecompounds Such physiologically acceptable compounds include, forexample, carbohydrates, such as glucose, sucrose or dextrans,antioxidants, such as ascorbic acid or glutathione, chelating agents,low molecular weight proteins or other stabilizers or excipients Oneskilled in the art would know that the choice of a pharmaceuticallyacceptable carrier, including a physiologically acceptable compound,depends, for example, on the route of administration of the compositionPharmaceutically acceptable adjuvants, buffering agents, dispersingagents, and the like, may also be incorporated into the pharmaceuticalcompositions.

The compounds can be administered parenterally. Preparations of thecompounds for parenteral administration must be sterile. Sterilizationis readily accomplished by filtration through sterile filtrationmembranes, optionally prior to or following lyophilization andreconstitution. The parenteral route for administration of compounds isin accord with known methods, e.g. injection or infusion by intravenous,intraperitoneal, intramuscular, intraarterial, or intralesional routes.The compounds may be administered continuously by infusion or by bolusinjection. A typical composition for intravenous infusion could be madeup to contain 100 to 500 ml of sterile 0.9% NaCl or 5% glucoseoptionally supplemented with a 20% albumin solution and 1 mg to 10 g ofthe compound, depending on the particular type of compound and itsrequired dosing regimen. Methods for preparing parenterallyadministrable compositions are well known in the art.

EXAMPLES Example 1 Generation of Anti-huNKp46 Antibodies

Balb/c mice were immunized with a recombinant human NKp46 extracellulardomain recombinant-Fc protein. Mice received one primo-immunization withan emulsion of 50 μg NKp46 protein and Complete Freund Adjuvant,intraperitoneally, a 2nd immunization with an emulsion of 50 μg NKp46protein and Incomplete Freund Adjuvant, intraperitoneally, and finally aboost with 10 μg NKp46 protein, intravenously. Immune spleen cells werefused 3 days after the boost with X63.Ag8.653 immortalized B cells, andcultured in the presence of irradiated spleen cells.

Primary screen: Supernatant (SN) of growing clones were tested in aprimary screen by flow cytometry using a cell line expressing the humanNKp46 construct at the cell surface. Briefly, for FACS screening, thepresence of reacting antibodies in supernatants was revealed by Goatanti-mouse polyclonal antibody (pAb) labeled with PE.

A selection of antibodies that bound NKp46 were selected, produced andtheir variable regions further evaluated for their activity in thecontext of a bispecific molecule.

Example 2 Identification of a Bispecific Antibody Format that Binds FcRnbut not FcγR for Targeting Effector Cell Receptors

The aim of this experiment was to develop a new bispecific proteinuseful in targeting receptors on immune effector cells and a secondantigen of interest, in which a monomeric Fc domain is placed on apolypeptide between two antigen binding domains. The proteins and bindsto the two antigens monovalently, which retains at least partial bindingto the human neonatal Fc receptor (FcRn) but does not substantially bindhuman CD16 and/or other human Fcγ receptors.

A first step was to produce a single chain protein with a monomeric Fcdomain as a single chain protein to investigate whether a monomeric Fcdomain would bind to the human neonatal Fc receptor (FcRn) and human Fcγreceptors.

Example 2-1 Construction and Binding Analysis ofAnti-CD19-IgG1-Fcmono-Anti-CD3

A bispecific Fc-based on a scFv specific for tumor antigen CD19(anti-CD19 scFv) and a scFV specific for activating receptor CD3 on a Tcell (anti-CD3 scFv) was used to assess FcRn binding and CD19-bindingfunctions of a new monomeric bispecific polypeptide format. The domainarrangement of the final polypeptide is shown in FIG. 2 and is alsoreferred to as the “F1” format in FIG. 6A (the star in the CH2 domainindicates an optional N297S mutation, not included in the polypeptidetested here).

A bispecific monomeric Fc-containing polypeptide was constructed basedon an scFv specific for the tumor antigen CD19 (anti-CD19 scFv) and anscFV specific for an activating receptor CD3 on a T cell (anti-CD3scFv). The CH3 domain incorporated the mutations (EU numbering) L351K,T366S, P395V, F405R, T407A and K409Y. The polypeptide has domainsarranged as follows: anti-CD19-CH2-CH3-anti-CD3. DNA sequence coding fora CH3/VH linker peptide having the amino acid sequence STGS was designedin order to insert a specific Sall restriction site at the CH3-VHjunction.

The CH3 domain incorporated the mutations (EU numbering) L351K, T366S,P395V, F405R, T407A and K409Y. The CH2 domain was a wild-type CH2. DNAand amino acid sequences for the monomeric CH2-CH3 Fc portion and theanti-CD19 are shown below.

The light chain and heavy chain DNA and amino acid sequences for theanti-CD19 scFv were as follows:

Anti-CD1 9-VK (SEQ ID NO: 3) GACATTCAGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCA AGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATTTGAACTGGTACCAACAGATACCAGGACAGCC ACCCAAACTCCTCATCTATGATGCATCCAATCTAGTATCTGGGATTCCACCCAGGTTTAGTGGCAGTGGG TCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGAAGGTGGATGCTGCAACCTATCACTGTCAGC AAAGTACTGAGGACCCTTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA Anti-CD1 9-VK (SEQ ID NO: 4)DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDS YLNWYQQIPGQPPKLLIYDASNLVSGIPPRESGSGSGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGG TKLEIK Anti-CD1 9-VH (SEQ ID NO: 5)CAGGTTCAGCTGCAGCAGTCTGGGGCTGAGCTGGT GCGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAAGCATCTGGCTACGCATTCAGTAGCTACTGGATGAAC TGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACAGATTTGGCCTGGAGATGGTGATACTA ACTACAACGGAAAGTTCAAGGGCAAGGCCACACTGACTGCAGACGAATCCTCCAGCACAGCCTACATGCA GCTCAGCAGCCTGGCCTCTGAGGACTCTGCGGTCTATTTCTGTGCAAGACGAGAAACGACCACTGTCGGG CGTTATTACTATGCTATGGACTACTGGGGTCAAGGAACCACAGTCACCGTCTCCTCA Anti-CD1 9-VH (SEQ ID NO: 6)QVQLQQSGAELVRPGSSVKISCKASGYAFSSYWMN WVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLSSLASEDSAVYFCARRETTTVG RYYYAMDYWGQGTTVTVSS

The DNA and amino acid sequences for the monomeric CH2-CH3 Fc portionand final bispecific polypeptide were as follows:

IgG1-Fcmono (the last K was removed in that construct) (SEQ ID NO: 7)GCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCT CTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGAC GTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGA CAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGA CTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACC ATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCAAGCCCCCATCCCGGGAGGAGATGA CCAAGAACCAGGTCAGCCTGTCCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGA GAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGGTTCCCGTGCTGGACTCCGACGGCTCCTTCCGC CTCGCTAGCTACCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCCCCGGGG IgG1-Fcmono* (*the last K residuewas removed in that construct) (SEQ ID NO: 8)APELLGGPSVFLFPPKPKDTLMISRTPEVICVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKT ISKAKGQPREPQVYTKPPSREEMTKNQVSLSCLVKGFYPSDIAVEWESNGQPENNYKTTVPVLDSDGSFR LASYLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG Anti-CD19-F1-Anti-CD3 Complete sequence (mature protein)(SEQ ID NO: 9) DIQLTQSPASLAVSLGQRATISCKASQSVDYDGDSYLNWYQQIPGQPPKLLIYDASNLVSGIPPRFSGSG SGTDFTLNIHPVEKVDAATYHCQQSTEDPWTFGGGTKLEIKGGGGSGGGGSGGGGSQVQLQQSGAELVRP GSSVKISCKASGYAFSSYWMNWVKQRPGQGLEWIGQIWPGDGDTNYNGKFKGKATLTADESSSTAYMQLS SLASEDSAVYFCARRETTTVGRYYYAMDYWGQGTTVTVSSGGGSSAPELLGGPSVFLFPPKPKDTLMISR TPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTKPPSREEMTKNQVSLSCLVKGFYPSDIAVEWESNGQPENNYKTTV PVLDSDGSFRLASYLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGSTGSDIKLQQSGAELARPG ASVKMSCKTSGYTFTRYTMHWVKQRPGQGLEWIGYINPSRGYTNYNQKFKDKATLTTDKSSSTAYMQLSS LTSEDSAVYYCARYYDDHYCLDYWGQGTTLTVSSVEGGSGGSGGSGGSGGVDDIQLTQSPAIMSASPGEK VTMTCRASSSVSYMNWYQQKSGTSPKRWIYDTSKVASGVPYRFSGSGSGTSYSLTISSMEAEDAATYYCQ QWSSNPLTFGAGTKLELK 

Cloning and Production of the Recombinant Proteins

Coding sequences were generated by direct synthesis and/or by PCR. PCRwere performed using the PrimeSTAR MAX DNA polymerase (Takara, #R045A)and PCR products were purified from 1% agarose gel using the NucleoSpingel and PCR clean-up kit (Macherey-Nagel, #740609.250). Once purifiedthe PCR product were quantified prior to the In-Fusion ligation reactionperformed as described in the manufacturer's protocol (ClonTech,#ST0345). The plasmids were obtained after a miniprep preparation run onan EVO200 (Tecan) using the Nucleospin 96 plasmid kit (Macherey-Nagel,#740625.4). Plasmids were then sequenced for sequences confirmationbefore to transfecting the CHO cell line.

CHO cells were grown in the CD-CHO medium (Invitrogen) complemented withphenol red and 6 mM GlutaMax. The day before the transfection, cells arecounted and seeded at 175.000 cells/ml. For the transfection, cells(200.000 cells/transfection) are prepared as described in the AMAXA SFcell line kit (AMAXA, #V4XC-2032) and nucleofected using the DS137protocol with the Nucleofector 4D device. All the tranfections wereperformed using 300 ng of verified plasmids. After transfection, cellsare seeded into 24 well plates in pre-warmed culture medium. After 24H,hygromycine B was added in the culture medium (200 μg/ml). Proteinexpression is monitored after one week in culture. Cells expressing theproteins are then sub-cloned to obtain the best producers. Sub-cloningwas performed using 96 flat-bottom well plates in which the cells areseeded at one cell per well into 200 μl of culture medium complementedwith 200 μg/ml of hygromycine B. Cells were left for three weeks beforeto test the clone's productivity.

Recombinant proteins which contain a IgG1-Fc fragment are purified usingProtein-A beads (-rProteinA Sepharose fast flow, GE Healthcare, ref.:17-1279-03). Briefly, cell culture supernatants were concentrated,clarified by centrifugation and injected onto Protein-A columns tocapture the recombinant Fc containing proteins. Proteins were eluted atacidic pH (citric acid 0.1M pH3), immediately neutralized using TRIS-HCLpH8.5 and dialyzed against 1×PBS. Recombinant scFv which contain a “sixhis” tag were purified by affinity chromatography using Cobalt resin.Other recombinant scFv were purified by size exclusion chromatography(SEC).

Example 2-2: Binding Analysis of Anti-CD19-IgG1-Fcmono-Anti-CD3 to B221,JURKAT, HUT78 and CHO Cell Lines

Cells were harvested and stained with the cell supernatant of theanti-CD19-F1-anti-CD3 producing cells during 1 H at 4° C. After twowashes in staining buffer (PBS1X/BSA 0.2%/EDTA 2 mM), cells were stainedfor 30 min at 4° C. with goat anti-human (Fc)-PE antibody (1M0550Beckman Coulter-1/200). After two washes, stainings were acquired on aBD FACS Canto II and analyzed using the FlowJo software.

CD3 and CD19 expression were also controlled by flow cytometry: Cellswere harvested and stained in PBS1X/BSA 0.2%/EDTA 2 mM buffer during 30min at 4° C. using 5 μl of the anti-CD3-APC and 5 μl of theanti-CD19-FITC antibodies. After two washes, stainings were acquired ona BD FACS Canto II and analyzed using the FlowJo software.

The Anti-CD19-F1-Anti-CD3 protein binds to the CD3 cell lines (HUT78 andJURKAT cell lines) and the CD19 cell line (B221 cell line) but not tothe CHO cell line used as a negative control.

Example 2-3 T- and B-Cell Aggregation by Purified Anti-CD19-F1-Anti-CD3

Purified Anti-CD19-F1-Anti-CD3 was tested in a T/B cell aggregationassay to evaluate whether the antibody is functional in bringingtogether CD19 and CD3 expressing cells.

Results are shown in FIG. 4. The top panel shows thatAnti-CD19-F1-Anti-CD3 does not cause aggregation in the presence of B221(CD19) or JURKAT (CD3) cell lines, but it does cause aggregation ofcells when both B221 and JURKAT cells are co-incubated, illustratingthat the bispecific antibody is functional. The lower panel showscontrol without antibody.

Example 2-4 Binding of Bispecific Monomeric Fc Polypeptide to FcRnAffinity Study by Surface Plasmon Resonance (SPR) Biacore T100 GeneralProcedure and Reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments Acetate Buffer (50 mMAcetate pH5.6, 150 mM NaCl, 0.1% surfactant p20) and HBS-EP+ (Biacore GEHealthcare) served as running buffer and regeneration bufferrespectively. Sensorgrams were analyzed with Biacore T100 Evaluationsoftware. Recombinant mouse FcRn was purchase from R&D Systems.

Immobilization of FcRn

Recombinant FcRn proteins were immobilized covalently to carboxyl groupsin the dextran layer on a Sensor Chip CM5. The chip surface wasactivated with EDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl)carbodiimidehydrochloride and N-hydroxysuccinimide (Biacore GEHealthcare)). FcRn proteins were diluted to 10 μg/ml in coupling buffer(10 mM acetate, pH 5.6) and injected until the appropriateimmobilization level was reached (i.e. 2500 RU). Deactivation of theremaining activated groups was performed using 100 mM ethanolamine pH 8(Biacore GE Healthcare).

Affinity Study

Monovalent affinity study was done following the Single Cycle Kinetic(SCK) protocol. Five serial dilutions of soluble analytes (antibodiesand bi-specific molecules) ranging from 41.5 to 660 nM were injectedover the FcRn (without regeneration) and allowed to dissociate for 10min before regeneration. For each analyte, the entire sensorgram wasfitted using the 1:1 SCK binding model.

Results

Anti-CD19-F1-Anti-CD3 having its CH2-CH3 domains placed between twoantigen binding domains, here two scFv, was evaluated to assess whethersuch bispecific monomeric Fc protein could retain binding to FcRn andthereby have improved in vivo half-lives compared to conventionbispecific antibodies. Results showed that FcRn binding was retained,the model suggesting 1:1 ratio (1 FcRn for each monomeric Fc) instead of2:1 ration (2 FcRn for each antibody) for a regular IgG. Results areshown in FIG. 5.

Affinity was evaluated using SPR, in comparison to a chimeric fulllength antibody having human IgG1 constant regions. Results are shown inFIG. 5. The monomeric Fc retained significant monomeric binding to FcRn(monomeric Fc: affinity of KD=194 nM; full length antibody with bivalentbinding: avidity of KD=15.4 nM).

Example 3 Construction of Multimeric Bispecific Monomeric Polypeptides

The aim of this experiment was to develop a new bispecific proteinformat that has advantages in production over currently availablebispecific antibodies in development, e.g. DART and BITE antibodies.

The initial bispecific protein was designed to place an Fc domain on apolypeptide together with a binding domain that bind to a receptorpresent on the surface of immune effector cells, chosen to be anti-NKp46which binds the activating receptor NKp46 on NK cells, and ananti-target antigen binding domain, chosen to be anti-CD19 which bindsthe lymphoma tumor antigen. The bispecific protein binds to NKp46monovalently while the monomeric Fc domain retains at least partialbinding to the human neonatal Fc receptor (FcRn), yet does notsubstantially bind human CD16 and/or other human Fcγ receptors.Consequently, the bispecific protein will not induce Fcγ-mediated (e.g.CD16-mediated) target cell lysis.

It was unknown what activating receptors on NK cells would contribute tolysis of target cells, and as anti-NKp46 antibodies may block NKp46,whether cytotoxicity could be mediated by NKp46 triggering. Weinvestigated whether the bispecific protein format could induce NKp46triggering, and moreover without inducing NKp46 agonism in the absenceof target cells, which could lead to inappropriate NK activation distantfrom the target and/or decreased overall activity toward target cells.

Multimeric proteins that comprise two or three polypeptide chains,wherein the Fc domain remains monomeric, were developed that arecompatible for use with antibody variable regions that do not maintainbinding to their target when converted to scFv format. The formats canbe used conveniently for antibody screening; by incorporating at leastone binding region as a F(ab) structure, any anti-target (e.g.anti-tumor) antibody variable region can be directly expressed in abispecific construct as the F(ab) format within the bispecific proteinand tested, irrespective of whether the antibody would retain binding asan scFv, thereby simplifying screening and enhancing the number ofantibodies available. These formats in which the Fc domain remainsmonomeric have the advantage of maintaining maximum conformationalflexibility which may permit optimal binding to both target antigens,e.g., effector cell receptor and/or target antigens.

Different constructs were made for use in the preparation of bispecificantibodies using the variable domains DNA and amino acid sequences fromthe scFv specific for tumor antigen CD19 described in Example 2-1, anddifferent variable regions from antibodies specific for the NKp46receptor identified in Example 1.

For the Fc domain to remain monomeric in single chain polypeptides ormultimers in which only one chain had an Fc domain, CH3-CH3 dimerizationwas prevented through two different strategies: (1) through the use ofCH3 domain incorporating the mutations (EU numbering) L351K, T366S,P395V, F405R, T407A and K409Y; or (2) through the use of a tandem CH3domain in which the tandem CH3 domains separated by a flexible linkerassociated with one another, in turn preventing interchain CH3-CH3dimerization. The DNA and amino acid sequences for the monomeric CH2-CH3Fc portion with point mutations were as in Example 2-1. The DNA andamino acid sequences for the monomeric CH2-CH3-linker-CH3 Fc portionwith tandem CH3 domains is shown in FIGS. 6A-6D.

The light chain and heavy chain DNA and amino acid sequences for theanti-CD19 scFv were as in Example 2-1. Proteins were cloned, producedand purified as in Example 2-1. Shown below are an exemplary light chainand heavy chain DNA and amino acid sequences for an anti-NKp46 scFvNKp46-3.

scFv anti- scFv sequence NKp46 (VHVK)/-stop NKp46-3STGSEVQLQQSGPELVKPGASVKIS CKTSGYTFTEYTMHWVKQSHGKSLEWIGGISPNIGGTSYNQKFKGKATLT VDKSSSTAYMELRSLTSEDSAVYYCARRGGSFDYWGQGTTLTVSSVEGGS GGSGGSGGSGGVDDIVMTQSPATLSVTPGDRVSLSCRASQSISDYLHWYQ QKSHESPRLLIKYASQSISGIPSRFSGSGSGSDFTLSINSVEPEDVGVYY CQNGHSFPLTFGAGTKLELK- (SEQ ID NO: 10)Format 1 (F1) (Anti-CD19-IgG1-Fcmono-Anti-NKp46 (scFv))

The domain structure of Format 1 (F1) is shown in FIG. 6A. A bispecificFc-containing polypeptide was constructed based on an scFv specific forthe tumor antigen CD19 (anti-CD19 scFv) and an scFV specific for theNKp46 receptor. The polypeptide is a single chain polypeptide havingdomains arranged (N- to C-terminal) as follows:(VK-VH)^(anti-CD19)-CH2-CH3-(VH-VK)^(anti-NKp46)

A DNA sequence coding for a CH3/VH linker peptide having the amino acidsequence STGS was designed in order to insert a specific Sallrestriction site at the CH3-VH junction. The domain arrangement of thefinal polypeptide in shown in FIG. 2 (star in the CH2 domain indicatesan optional N297S mutation). The (VK-VH) units include a linker betweenthe VH and VK domains. Proteins were cloned, produced and purified as inExample 2-1.

Format 2 (F2): CD19-F2-NKp46-3

The domain structure of F2 polypeptides is shown in FIG. 6A. The DNA andamino acid sequences for the monomeric CH2-CH3 Fc portion were as inExample 2-1 containing CH3 domain mutations (the mutations (EUnumbering) L351K, T366S, P395V, F405R, T407A and K409Y. The heterodimeris made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

(VK-VH)^(anti-CD19)-CH2-CH3-VH^(anti-NKp46)-CH1 and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CK.

The (VK-VH) unit was made up of a VH domain, a linker and a VK unit(i.e. an scFv). As with other formats of the bispecific polypeptides,the DNA sequence coded for a CH3/VH linker peptide having the amino acidsequence STGS designed in order to insert a specific Sall restrictionsite at the CH3-VH junction. Proteins were cloned, produced and purifiedas in Example 2-1. The amino acid sequences for the CD19-F2-NKp46-3Polypeptide chain 1 is shown in SEQ ID NO: 11 and CD19-F2-NKp46-3Polypeptide chain 2 in SEQ ID NO: 12.

Format 8 (F8)

The domain structure of F8 polypeptides is shown in FIG. 6B. The DNA andamino acid sequences for the monomeric CH2-CH3 Fc portion were as inFormat F2 containing CH3 domain mutations (the mutations (EU numbering)L351K, T366S, P395V, F405R, T407A and K409Y, as well as a N297S mutationto prevent N-linked glycosylation and abolish FcγR binding. Threevariants of F8 proteins were produced: (a) cysteine residues in thehinge region left intact (wild-type, referred to as F8A), (b) cysteineresidues in the hinge region replaced by serine residues (F8B), and (c)a linker sequence GGGSS replacing residues DKTHTCPPCP in the hinge(F8C). Variants F8B and F8C provided advantages in production byavoiding formation of homodimers of the central chain. The heterotrimeris made up of;

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

VH^(anti-CD19)-CH1-CH2-CH3-VH^(anti-NKp46)-CK and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CH1 and

(3) a third polypeptide chain having domains arranged as follows (N- toC-terminal):

VK^(anti-CD19)-CK

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 3.7 mg/L (F8C) andwith a simple SEC profile. The amino acid sequences for the three F8protein chains for the F8 “C” variant are shown in SEQ ID NOS 13, 14 and15.

Format 9 (F9): CD19-F9-NKp46-3

The F9 polypeptide is a trimeric polypeptide having a centralpolypeptide chain and two polypeptide chains each of which associatewith the central chain via CH1-CK dimerization. The domain structure ofthe trimeric F9 protein is shown in FIG. 6B, wherein the bonds betweenthe CH1 and CK domains are interchain disulfide bonds. The two antigenbinding domains have a F(ab) structure permitting the use of antibodiesirrespective of whether they remain functional in scFv format. The DNAand amino acid sequences for the CH2-CH3 Fc portion comprised a tandemCH3 domain in which the two CH3 domains on the same polypeptide chainassociated with one another, thereby preventing dimerization betweendifferent bispecific proteins. The CH2 domain included a N297Ssubstitution. Three variants of F9 proteins were produced: (a) cysteineresidues in the hinge region left intact (wild-type, referred to asF9A), (b) cysteine residues in the hinge region replaced by serineresidues (F9B), and (c) a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F9C). Variants F9B and F9C provided advantagesin production by avoiding formation of homodimers of the central chain.The heterotrimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

VH^(anti-CD19)-CH1-CH2-CH3-CH3-VH^(anti-NKp46)-CK and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CH1 and

(3) a third polypeptide chain having domains arranged as follows (N- toC-terminal):

VK^(anti-CD19)-CK

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 8.7 mg/L (F9A) and3.0 mg/L (F9B), and with a simple SEC profile. The amino acid sequencesfor the three F9 protein chains for each of variants F9A, F9B and F9Care shown in the SEQ ID NOS listed in the table below.

Protein SEQ ID NOS F9A 16, 17, 18 F9B 19, 20, 21 F9C 22, 23, 24

Format 10 (F10): CD19-F10-NKp46-3

The F10 polypeptide is a dimeric protein having a central polypeptidechain and a second polypeptide chain which associates with the centralchain via CH1-CK dimerization. The domain structure of the dimeric F10proteins is shown in FIG. 6B wherein the bonds between the CH1 and CKdomains are interchain disulfide bonds. One of the two antigen bindingdomains has a Fab structure, and the other is a scFv. The DNA and aminoacid sequences for the CH2-CH3 Fc portion comprised a tandem CH3 domainas in Format F9 and a CH2 domain with a N297S substitution.Additionally, three variants of F10 proteins were produced: (a) cysteineresidues in the hinge region left intact (wild-type, referred to asF10A), (b) cysteine residues in the hinge region replaced by serineresidues (F10B, and (c) a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F10C). Variants F10B an F10C providedadvantages in production by avoiding formation of homodimers of thecentral chain. The (VK-VH) unit was made up of a VH domain, a linker anda VK unit (scFv). The heterodimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

VH^(anti-CD19)-CH1-CH2-CH3-CH3-(VH-VK)^(anti-NKp46) and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a good production yield of 2 mg/L (F10A) andwith a simple SEC profile. The amino acid sequences for the three F9protein chains for each of variants F10A, F10B and F10C are shown in theSEQ ID NOS listed in the table below.

Protein SEQ ID NOS F10A 25, 26 F10B 27, 28 F10C 29, 30

Format 11 (F11): CD19-F11-NKp46-3

The domain structure of F11 polypeptides is shown in FIG. 6C. Theheterodimeric protein is similar to F10 but the structures of theantigen binding domains are reversed. One of the two antigen bindingdomains has a Fab-like structure, and the other is a scFv. Theheterodimer is made up of

-   -   (1) a first (central) polypeptide chain having domains arranged        as follows (N- to C-terminal):

(VK-VH)^(anti-CD19)-CH2-CH3-CH3-VH^(anti-NKp46)-CK and

-   -   (2) a second polypeptide chain having domains arranged as        follows (N- to C-terminal): VK^(anti-NKp46)-CH1.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a good production yield of 2 mg/L and with asimple SEC profile. The amino acid sequences for the two chains of theF11 protein are shown in SEQ ID NOS 31 and 32.

Format 12 (F12): CD19-F12-NKp46-3

The domain structure of the dimeric F12 polypeptides is shown in FIG.6C, wherein the bonds between the CH1 and CK domains are disulfidebonds. The heterodimeric protein is similar to F11 but the CH1 and CKdomains within the F(ab) structure are inversed. The heterodimer is madeup of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

(VK-VH)^(anti-CD19)-CH2-CH3-CH3-VH^(anti-NKp46)-CH1 and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-NKp46)-CK.

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a good production yield of 2.8 mg/L and with asimple SEC profile. The amino acid sequences for the two chains of theF12 protein are shown in SEQ ID NOS: 33 and 34.

Format 17 (F17): CD19-F17-NKp46-3

The domain structure of the trimeric F17 polypeptides is shown in FIG.6C, wherein the bonds between the CH1 and CK domains are disulfidebonds. The heterodimeric protein is similar to F9 but the VH and VKdomains, and the CH1 and CK, domains within the C-terminal F(ab)structure are each respectively inversed with their partner. Theheterotrimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

VH^(anti-CD19)-CH1-CH2-CH3-CH3-VK^(anti-NKp46)-CH1 and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VH^(anti-NKp46)-CK and

(3) a third polypeptide chain having domains arranged as follows (N- toC-terminal):

VK^(anti-CD19)-CK

Additionally, three variants of F17 proteins were produced: (a) cysteineresidues in the hinge region left intact (wild-type, referred to asF17A), (b) cysteine residues in the hinge region replaced by serineresidues (F10B, and (c) a linker sequence GGGSS replacing residuesDKTHTCPPCP in the hinge (F17C). Proteins were cloned, produced andpurified as in Example 2-1. The amino acid sequences for the threechains of the F17B protein chains are shown in SEQ ID NOS: 35, 36 and37.

Example 4 Bispecific NKp46 Antibody Formats with Dimeric Fc Domains

New protein constructions with dimeric Fc domains were developed thatshare advantages of the monomeric Fc domain proteins of Example 3 butbind to FcRn with greater affinity, but which also have low orsubstantially lack of binding to FcγR. The polypeptide formats weretested to investigate the functionality of heterodimeric proteinscomprising a VH-(CH1 or CK)-CH2-CH3- comprising chain and a VK-(CH1 orCK)-CH2-CH3- comprising chain. One of both of the CH3 domains will thenbe fused, optionally via intervening amino acid sequences or domains, toa variable domain(s) (a single variable domain that associates with avariable domain on a separated polypeptide chain, a tandem variabledomain (e.g., an scFv), or a single variable domain that is capable ofbinding antigen as a single variable domain. The two chains thenassociate by CH1-CK dimerization to form disulfide linked dimers, or ifassociated with a third chain, to form trimers.

Different constructs were made for use in the preparation of abispecific antibody using the variable domains DNA and amino acidsequences derived from the scFv specific for tumor antigen CD19described in Example 2-1 and different variable regions from antibodiesspecific for NKp46 identified in Example 1. Proteins were cloned,produced and purified as in Example 2-1. Domains structures are shown inFIGS. 6A-6D.

Format 5 (F5): CD19-F5-NKp46-3

The domain structure of the trimeric F5 polypeptide is shown in FIG. 6D,wherein the interchain bonds between hinge domains and interchain bondsbetween the CH1 and CK domains are interchain disulfide bonds. Theheterotrimer is made up of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

VH^(anti-CD19)-CH1-CH2-CH3-VH^(anti-NKp46)-CK and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK-CH2-CH3 and

(3) a third polypeptide chain having domains arranged as follows (N- toC-terminal):

VH^(anti-NKp46)-CH1

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 37 mg/L and with asimple SEC profile. The amino acid sequences of the three chains areshown in SEQ ID NOS: 38, 39 and 40.

Format 6 (F6): CD19-F6-NKp46-3

The domain structure of heterotrimeric F6 polypeptides is shown in FIG.6D. The F6 protein is the same as F5, but with a N297S substitution toavoid N-linked glycosylation. Proteins were cloned, produced andpurified as in Example 2-1. Bispecific proteins was purified from cellculture supernatant by affinity chromatography using prot-A beads andanalysed and purified by SEC. The protein showed a high production yieldof 12 mg/L and with a simple SEC profile. The amino acid sequences ofthe three chains of the F6 protein are shown in SEQ ID NOS: 41, 42 and43.

Format 7 (F7): CD19-F7-NKp46-3

The domain structure of heterotrimeric F7 polypeptides is shown in FIG.6D. The F7 protein is the same as F6, but with cysteine to serinesubstitutions in the CH1 and CK domains that are linked at theirC-termini to the Fc domains, to prevent formation of a minor populationof dimeric species of the central chain with the VK^(anti-NKp46)-CH1chain. Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 11 mg/L and with asimple SEC profile. The amino acid sequences of the three chains of the76 protein are shown in SEQ ID NOS: 44, 45 and 46.

Format 13 (F13): CD19-F13-NKp46-3

The domain structure of the dimeric F13 polypeptide is shown in FIG. 6D,wherein the interchain bonds between hinge domains (indicated betweenCH1/CK and CH2 domains on a chain) and interchain bonds between the CH1and CK domains are interchain disulfide bonds. The heterodimer is madeup of:

(1) a first (central) polypeptide chain having domains arranged asfollows (N- to C-terminal):

VH^(anti-CD19)-CH1-CH2-CH3-(VH-VK)^(anti-NKp46) and

(2) a second polypeptide chain having domains arranged as follows (N- toC-terminal): VK^(anti-CD19)-CK-CH2-CH3.

The (VH-VK) unit was made up of a VH domain, a linker and a VK unit(scFv).

Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 6.4 mg/L and with asimple SEC profile. The amino acid sequences of the two chains of theF13 protein are shown in SEQ ID NOS: 47 and 48.

Format 14 (F14): CD19-F14-NKp46-3

The domain structure of the dimeric F14 polypeptide is shown in FIG. 6E.The F14 polypeptide is a dimeric polypeptide which shares the structureof the F13 format, but instead of a wild-type Fc domain (CH2-CH3), theF14 has CH2 domain mutations N297S to abolish N-linked glycosylation.Proteins were cloned, produced and purified as in Example 2-1.Bispecific proteins was purified from cell culture supernatant byaffinity chromatography using prot-A beads and analysed and purified bySEC. The protein showed a high production yield of 2.4 mg/L and with asimple SEC profile. The amino acid sequences of the two chains of theF14 protein are shown in SEQ ID NOS: 49 and 50.

Format 15 (F15): CD19-F15-NKp46-3

The domain structure of the trimeric F15 polypeptides is shown in FIG.6E. The F15 polypeptide is a dimeric polypeptide which shares thestructure of the F6 format, but differs by inversion of the N-terminalVH-CH1 and VK-CK units between the central and second chains. Proteinswere cloned, produced and purified as in Example 2-1. Bispecificproteins was purified from cell culture supernatant by affinitychromatography using prot-A beads and analysed and purified by SEC. Theprotein showed a good production yield of 0.9 mg/L and with a simple SECprofile. The amino acid sequences of the three chains of the F15 proteinare shown in SEQ ID NOS: 51, 52 and 53.

Format 16 (F16): CD19-F16-NKp46-3

The domain structure of the trimeric F16 polypeptide is shown in FIG.6E. The F16 polypeptide is a dimeric polypeptide which shares thestructure of the F6 format, but differs by inversion of the C-terminalVH-CK and VK-CH1 units between the central and second chains. Proteinswere cloned, produced and purified as in Example 2-1. The amino acidsequences of the three chains of the F16 protein are shown in SEQ IDNOS: 54, 55 and 56.

Example 5 NKp46 Binding Affinity by Bispecific Proteins by SurfacePlasmon Resonance (SPR) Biacore T100 General Procedure and Reagents

SPR measurements were performed on a Biacore T100 apparatus (Biacore GEHealthcare) at 25° C. In all Biacore experiments HBS-EP+(Biacore GEHealthcare) and NaOH 10 mM served as running buffer and regenerationbuffer respectively. Sensorgrams were analyzed with Biacore T100Evaluation software. Protein-A was purchase from (GE Healthcare). HumanNKp46 recombinant proteins were cloned, produced and purified at InnatePharma.

Immobilization of Protein-A

Protein-A proteins were immobilized covalently to carboxyl groups in thedextran layer on a Sensor Chip CM5. The chip surface was activated withEDC/NHS (N-ethyl-N′-(3-dimethylaminopropyl) carbodiimidehydrochlorideand N-hydroxysuccinimide (Biacore GE Healthcare)). Protein-A was dilutedto 10 μg/ml in coupling buffer (10 mM acetate, pH 5.6) and injecteduntil the appropriate immobilization level was reached (i.e. 2000 RU).Deactivation of the remaining activated groups was performed using 100mM ethanolamine pH 8 (Biacore GE Healthcare).

Binding Study

Antibodies were tested as different formats F5, F6, F9, F10, F11, F13,F14 and compared to the single chain format (F1), and an anti-NKp46antibody as a full-length human IgG1.

Bispecific proteins at 1 μg/mL were captured onto Protein-A chip andrecombinant human NKp46 proteins were injected at 5 μg/mL over capturedbispecific antibodies. For blank subtraction, cycles were performedagain replacing NKp46 proteins with running buffer.

Affinity Study

Monovalent affinity study was done following a regular Capture-Kineticprotocol recommended by the manufacturer (Biacore GE Healthcare kineticwizard). Seven serial dilutions of human NKp46 recombinant proteins,ranging from 6.25 to 400 nM were sequentially injected over the capturedBi-Specific antibodies and allowed to dissociate for 10 min beforeregeneration. The entire sensorgram sets were fitted using the 1:1kinetic binding model.

Results

SPR showed that the bispecific polypeptides of formats F1, F5, F6, F9,F10, F11, F13, F14 retained binding to NKp46.

Monovalent affinities and kinetic association and dissociation rateconstants are shown below in the table 3 below.

TABLE 3 Bispecific mAb ka (1/Ms) kd (1/s) KD (M) CD19-F1-NKp46-3 7.05E+04 6.44E−04 9.14E−09  CD19-F5-NKp46-3 7.555E+4 0.00510 67E−09CD19-F6-NKp46-3 7.934E+4 0.00503 63E−09 CD19-F9A-NKp46-3 2.070E+50.00669 32E−09 CD19-F10A-NKp46-3 2.607E+5 0.00754 29E−09CD19-F11A-NKp46-3 3.388E+5 0.01044 30E−09 CD19-F13-NKp46-3 8.300E+40.00565 68E−09 CD19-F14-NKp46-3 8.826E+4 0.00546 62E−09 NKp46-3 IgG12.224E+5 0.00433 20E−09

Example 6 Engagement of NK Cells Against Daudi Tumor Target withFc-Containing NKp46 x CD19 Bispecific Protein

Bispecific antibodies having a monomeric Fc domain and a domainarrangement according to the single chain F1 or dimeric F2 formatsdescribed in Example 3, and a NKp46 binding region based on differentanti-NKp46 variable domains (NKp46-1, NKp46-2, NKp46-3 or NKp46-4) weretested for functional ability to direct NK cells to lyse CD19-positivetumor target cells (Daudi, a well characterized B lymphoblast cellline). The F2 proteins additionally included NKp46-9 variable regionswhich lost binding to NKp46 in the scFv format but which retainedbinding in the F(ab)-like format of F2.

Briefly, the cytolytic activity of each of (a) resting human NK cells,and (b) human NK cell line KHYG-1 transfected with human NKp46, wasassessed in a classical 4-h ⁵¹Cr-release assay in U-bottom 96 wellplates. Daudi cells were labelled with ⁵¹Cr (50 μCi (1.85 MBq)/1×10⁶cells), then mixed with KHYG-1 transfected with hNKp46 at aneffector/target ratio equal to 50 for KHYG-1, and 10 (for F1 proteins)or 8.8 (for F2 proteins) for resting NK cells, in the presence ofmonomeric bi-specific antibodies at different concentrations. Afterbrief centrifugation and 4 hours of incubation at 37° C., samples ofsupernatant were removed and transferred into a LumaPlate (Perkin ElmerLife Sciences, Boston, Mass.), and ⁵¹Cr release was measured with aTopCount NXT beta detector (PerkinElmer Life Sciences, Boston, Mass.).All experimental conditions were analyzed in triplicate, and thepercentage of specific lysis was determined as follows: 100×(mean cpmexperimental release−mean cpm spontaneous release)/(mean cpm totalrelease−mean cpm spontaneous release). Percentage of total release isobtained by lysis of target cells with 2% Triton X100 (Sigma) andspontaneous release corresponds to target cells in medium (withouteffectors or Abs).

Results

In the KHYG-1 hNKp46 NK experimental model, each bi-specific antibodyNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 induced specific lysis ofDaudi cells by human KHYG-1 hNKp46 NK cell line compared to negativecontrols (Human IgG1 isotype control (IC) and CD19/CD3 bi-specificantibodies), thereby showing that these antibodies induce Daudi targetcell lysis by KHYG-1 hNKp46 through CD19/NKp46 cross-linking.

When resting NK cells were used as effectors, each bi-specific antibodyNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 again induced specificlysis of Daudi cells by human NK cells compared to negative control(Human IgG1 isotype control (IC) antibody), thereby showing that theseantibodies induce Daudi target cell lysis by human NK cells throughCD19/NKp46 cross-linking. Rituximab (RTX, chimeric IgG1) was used as apositive control of ADCC (Antibody-Dependent Cell Cytotoxicity) byresting human NK cells. The maximal response obtained with RTX (at 10μg/ml in this assay) was 21.6% specific lysis illustrating that thebispecific antibodies have high target cell lysis activity. Results forexperiments with resting NK cells are shown in FIG. 7A for the singlechain F1 proteins and 7B for the dimeric F2 proteins.

Example 7 Comparison with Full Length Anti-NKp46 mAbs and DepletingAnti-Tumor mAbs: Only NKp46 x CD19 Bispecific Proteins PreventNon-Specific NK Activation

These studies aimed to investigate whether bispecific antibodies canmediate NKp46-mediated NK activation toward cancer target cells withouttriggering non-specific NK cell activation.

NKp46 x CD19 bispecific proteins having an arrangement according to theF2 format described in Example 3 with anti-NKp46 variable domains fromNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 were compared to:

(a) full-length monospecific anti-NKp46 antibodies (NKp46-3 as humanIgG1), and

(b) the anti-CD19 antibody as a full-length human IgG1 as ADCC inducingantibody control comparator.

The experiments further included as controls: rituximab, an anti-CD20ADCC inducing antibody control for a target antigen with high expressionlevels; anti-CD52 antibody alemtuzumab, a human IgG1, binds CD52 targetpresent on both targets and NK cells; and negative control isotypecontrol therapeutic antibody (a human IgG1 that does not bind a targetpresent on the target cells (HUG1-IC).

The different proteins were tested for functional effect on NK cellactivation in the presence of CD19-positive tumor target cells (Daudicells), in the presence of CD19-negative, CD16-positive target cells(HUT78 T-lymphoma cells), and in the absence of target cells.

Briefly, NK activation was tested by assessing CD69 and CD107 expressionon NK cells by flow cytometry. The assay was carried out in 96 U wellplates in completed RPMI, 150 μL final/well. Effector cells were freshNK cells purified from donors. Target cells were Daudi (CD19-positive),HUT78 (CD19-negative) or K562 (NK activation control cell line). Inaddition to K562 positive control, three conditions were tested, asfollows:

-   -   NK cell alone    -   NK cells vs Daudi (CD19+)    -   NK cells vs HUT78 (CD19−)

Effector:Target (E:T) ratio was 2.5:1 (50 000 E:20 000 T), with anantibody dilution range starting to 10 μg/mL with 1/4 dilution (n=8concentrations). Antibodies, target cells and effector cells were mixed;spun 1 min at 300 g; incubated 4 h at 37° C.; spun 3 min at 500 g;washed twice with Staining Buffer (SB); added 50 μL of staining Ab mix;incubated 30 min at 300 g; washed twice with SB resuspended pellet withCellFix; stored overnight at 4° C.; and fluorescence revealed with CantoII (HTS).

Results

1. NK Cells Alone

Results are shown in FIG. 8A. In the absence of target-antigenexpressing cells, none of the bispecific anti-NKp46 x anti-CD19 antibody(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9variable regions) activated NK cells as assessed by CD69 or CD107expression. Full-length anti-CD19 also did not activate NK cells.However, the full-length anti-NKp46 antibodies caused detectableactivation of NK cells. Alemtuzumab also induced activation of NK cells,at a very high level. Isotype control antibody did not induceactivation.

2. NK Cells Vs Daudi (CD19+)

Results are shown in FIG. 8B. In the presence of target-antigenexpressing cells, each of the bispecific anti-NKp46 x anti-CD19antibodies (including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 andNKp46-9 binding domains) activated NK cells. Full-length anti-CD19showed at best only very low activation of NK cells. Neither full-lengthanti-NKp46 antibodies or alemtuzumab showed substantial increase inactivation beyond what was observed in presence of NK cells alone. FIG.8B shows full-length anti-NKp46 antibodies showed a similar level ofbaseline activation observed in presence of NK cells alone. Alemtuzumabalso induced activation of NK cells a similar level of activationobserved in presence of NK cells alone, and at higher antibodyconcentrations in this setting (ET 2.5:1) the activation was greaterthan with the bispecific anti-NKp46 x anti-CD19 antibody. Isotypecontrol antibody did not induce activation.

3. NK Cells Vs HUT78 (CD19−)

Results are shown in FIG. 8C. In the presence of target-antigen-negativeHUT78 cells, none of the bispecific anti-NKp46 x anti-CD19 antibody(including each of the NKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9variable regions) activated NK cells. However, the full-lengthanti-NKp46 antibodies and alemtuzumab caused detectable activation of NKcells at a similar level observed in presence of NK cells alone. Isotypecontrol antibody did not induce activation.

In conclusion, the bispecific anti-NKp46 proteins are able to activateNK cells in a target-cell specific manner, unlike full-lengthmonospecific anti-NKp46 antibodies and full-length antibodies ofdepleting IgG isotypes which also activate NK cells in the absence oftarget cells. The NK cell activation achieved with anti-NKp46 bispecificproteins was higher than that observed with full length anti-CD19 IgG1antibodies.

Example 8 Comparative Efficacy with Depleting Anti-Tumor mAbs: NKp46 xCD19 Bispecific Proteins at Low ET Ratio

These studies aimed to investigate whether bispecific antibodies canmediate NKp46-mediated NK cell activation toward cancer target cells atlower effector:target ratios. The ET ratio used in this Example was 1:1which is believed to be closer to the setting that would be encounteredin vivo than the 2.5:1 ET ratio used in Example 7 or the 10:1 ET ratioof Example 6.

NKp46 x CD19 bispecific proteins having an arrangement according to theF2 format described in Example 3 with anti-NKp46 variable domains fromNKp46-1, NKp46-2, NKp46-3, NKp46-4 or NKp46-9 were compared to:

(a) full-length monospecific anti-NKp46 antibodies (NKp46-3 as humanIgG1), and

(b) the anti-CD19 antibody as a full-length human IgG1 as ADCC inducingantibody control comparator.

The experiments further included as controls: rituximab (an anti-CD20ADCC inducing antibody control for a target antigen with high expressionlevels); anti-CD52 antibody alemtuzumab (a human IgG1, binds CD52 targetpresent on both targets and NK cells), and negative control isotypecontrol therapeutic antibody (a human IgG1 that does not bind a targetpresent on the target cells (HUG1-IC). The different proteins weretested for functional effect on NK cell activation as assessed by CD69or CD107 expression in the presence of CD19-positive tumor target cells(Daudi cells), in the presence of CD19-negative, CD16-positive targetcells (HUT78 T-lymphoma cells), and in the absence of target cells. Theexperiments were carried out as in Example 7 except that the ET ratiowas 1:1.

Results

Results are shown in FIG. 9 (top panel: CD107 and bottom panel: CD69).In the presence of target-antigen expressing cells, each of thebispecific anti-NKp46 x anti-CD19 antibody (including each of theNKp46-1, NKp46-2, NKp46-3, NKp46-4 and NKp46-9 variable regions)activated NK cells in the presence of Daudi cells.

The activation induced by bispecific anti-NKp46 x anti-CD19 antibody inthe presence of Daudi cells was far more potent than the full-lengthhuman IgG1 anti-CD19 antibody as ADCC inducing antibody which had lowactivity in this setting. Furthermore, in this low E:T ratio setting theactivation induced by bispecific anti-NKp46 x anti-CD19 antibody was aspotent as anti-CD20 antibody rituximab, with a difference being observedonly at the highest concentrations that were 10 fold higher thanconcentrations in which differences were observed at the 2.5:1 ET ratio.

In the absence of target cells or in the in the presence of targetantigen-negative HUT78 cells, full-length anti-NKp46 antibodies andalemtuzumab showed a similar level of baseline activation observed inthe presence of Daudi cells. anti-NKp46 x anti-CD19 antibody did notactivate NK cells in presence of HUT78 cells.

In conclusion, the bispecific proteins are able to activate NK cells ina target-cell specific manner and at lower effector:target ratio aremore effective in mediating NK cell activation that traditional humanIgG1 antibodies.

Example 9 Mechanism of Action Studies

NKp46 x CD19 bispecific proteins having an arrangement according to theF2, F3 (a single chain format), F5 or F6 formats described in Examples 3or 4 with anti-NKp46 variable domains from NKp46-3 were compared torituximab (anti-CD20 ADCC inducing antibody), and a human IgG1 isotypecontrol antibody for functional ability to direct CD16-/NKp46+NK celllines to lyse CD19-positive tumor target cells.

Briefly, the cytolytic activity of the CD16-/NKp46+ human NK cell lineKHYG-1 was assessed in a classical 4-h ⁵¹Cr-release assay in U-bottom 96well plates. Daudi or B221 cells were labelled with ⁵¹Cr (50 μCi (1.85MBq)/1×10⁶ cells), then mixed with KHYG-1 at an effector/target ratioequal to 50:1, in the presence of test antibodies at dilution rangestarting from 10⁻⁷ mol/L with 1/5 dilution (n=8 concentrations)

After brief centrifugation and 4 hours of incubation at 37° C., 50 μL ofsupernatant were removed and transferred into a LumaPlate (Perkin ElmerLife Sciences, Boston, Mass.), and ⁵¹Cr release was measured with aTopCount NXT beta detector (PerkinElmer Life Sciences, Boston, Mass.).All experimental conditions were analyzed in triplicate, and thepercentage of specific lysis was determined as follows: 100×(mean cpmexperimental release−mean cpm spontaneous release)/(mean cpm totalrelease−mean cpm spontaneous release). Percentage of total release isobtained by lysis of target cells with 2% Triton X100 (Sigma) andspontaneous release corresponds to target cells in medium (withouteffectors or Abs).

Results

Results are shown in FIG. 10A (KHYG-1 vs Daudi) or 10B (KHYG-1 vs B221).In the KHYG-1 hNKp46 NK experimental model, each NKp46 x CD19 bispecificprotein (Format F2, F3, F5 and F6) induced specific lysis of Daudi orB221 cells by human KHYG-1 hNKp46 NK cell line, while rituximab andhuman IgG1 isotype control (IC) antibodies did not.

Example 10 Binding of Different Bispecific Formats to FcRn

Affinity of different antibody formats for human FcRn was studied bySurface Plasmon Resonance (SPR) by immobilizing recombinant FcRnproteins covalently to carboxyl groups in the dextran layer on a SensorChip CM5, as described in Example 2-5.

A chimeric full length anti-CD19 antibody having human IgG1 constantregions and NKp46 x CD19 bispecific proteins having an arrangementaccording to the F5, F6, F9, F10, F11, F13 or F14 formats described inExamples 3 or 4 with anti-NKp46 variable domains from NKp46-3 (NKp46-2for F2) were tested; for each analyte, the entire sensorgram was fittedusing the steady state or 1:1 SCK binding model.

Results are shown in the table below. The bispecific proteins havingdimeric Fc domains (formats F5, F6, F13, F14) bound to FcRn withaffinity similar to that of the full-length IgG1 antibody. Thebispecific proteins with monomeric Fc domains (F9, F10, F11) alsodisplayed binding to FcRn, however with lower affinity that thebispecific proteins having dimeric Fc domains.

Antibody/Bispecific SPR method KD nM Human IgG1/K Anti- SCK/Two state7.8 CD19 reaction CD19-F5-NKp46-3 SCK/Two state 2.6 reaction CD19-F6-NKp46-3 SCK/Two state 6.0 reaction CD19-F13- NKp46-3 SCK/Two state 15.2reaction CD19-F14- NKp46-3 SCK/Two state 14.0 reaction CD19-F9A- NKp46-3Steady State 858.5 CD19-F10A- NKp46-3 Steady State 432.8 CD19-F11-NKp46-3 Steady State 595.5

Example 11 Binding of Anti-CD19 x Anti-NKp46 to FcvR

Anti-CD19-F1-Anti-NKp46 having its CH2-CH3 domains placed between twoantigen binding domains, here two scFv, was evaluated to assess whethersuch bispecific monomeric Fc protein could retain binding to Fcγreceptors.

Human IgG1 antibodies and CD19/NKp46-1 bi-specific antibodies wereimmobilized onto a CM5 chip. Recombinant FcγRs (cynomolgus monkey andhuman CD64, CD32a, CD32b, and CD16) were cloned, produced and purifiedat Innate Pharma. FIG. 11 shows superimposed sensorgrams showing thebinding of Macaca fascicularis recombinant FcgRs (upper panels; CyCD64,CyCD32a, CYCD32b, CyCD16) and of Human recombinant FcgRs (lower panels;HuCD64, HuCD32a, HuCD32b, HuCD16a) to the immobilized human IgG1 control(grey) and CD19/NKp46-1 bi-specific antibody (black). Sensorgrams werealigned to zero in the y and x axis at the sample injection start.

FIG. 11 shows that while full length wild type human IgG1 bound to allcynomolgus and human Fcγ receptors, the CD19/NKp46-1 bi-specificantibodies did not bind to any of the receptors.

Example 12 Improved Product Profile and Yield of Different BispecificFormats Compared to Existing Formats

Blinatumomab and two bispecific antibodies having NKp46 and CD19 bindingregions based on F1 to F17 formats and NKp46-3, and blinatumomab,respectively were cloned and produced under format 6 (F6), DART and BITEformats following the same protocol and using the same expressionsystem. F6, DART and BITE bispecific proteins were purified from cellculture supernatant by affinity chromatography using prot-A beads for F6or Ni-NTA beads for DART and BITE. Purified proteins were furtheranalysed and purified by SEC (FIG. 12A). BITE and DART showed a very lowproduction yield compared to F6 and have a very complex SEC profile. Asshown in FIG. 12B (arrows), DART and BITE are barely detectable bySDS-PAGE after Coomassie staining in the expected SEC fractions (3 and 4for BITE and 4 and 5 for DART), whereas F6 format showed clear andsimple SEC and SDS-PAGE profiles with a major peak (fraction 3)containing the monomeric bispecific proteins. The major peak for the F6format corresponded to about 30% of the total proteins. Theseobservations are also true for F1 to F17 proteins (data not shown)indicating that the Fc domain (or Fc-derive domain) present in thoseformats facilitate the production and improve the quality and solubilityof bispecific proteins.

Moreover, the Fc domains present in proteins F1 to F17 have theadvantage of being adapted to affinity chromatography without the needfor incorporation of peptide tags that will thereafter remain present asan unwanted part of a therapeutic product, such as in the case of BiTeand DART antibodies which cannot be purified by protein A. F1 to F17antibodies are all bound by protein A. The table below showsproductivity of different formats.

SDS PAGE Final Format SEC Reduced Non Reduced « productivity » yield F5✓ ✓ ✓ 37 mg/L F6 ✓ ✓ ✓ 12 mg/L F7 ✓ ✓ ✓ 11 mg/L F8C ✓ ✓ ✓ 3.7 mg/L F9A ✓✓ ✓ 8.7 mg/L F9B ✓ ✓ ✓ 3.0 mg/L F10A ✓ ✓ ✓ 2.0 mg/L F11 ✓ ✓ ✓ 2.0 mg/LF12 ✓ ✓ ✓ 2.8 mg/L F13 ✓ ✓ ✓ 6.4 mg/L F14 ✓ ✓ ✓ 2.4 mg/L F15 ✓ ✓ ✓ 0.9mg/L BiTe — — — — DART — — — —

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way. Anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context. Recitation ofranges of values herein are merely intended to serve as a shorthandmethod of referring individually to each separate value falling withinthe range, unless otherwise indicated herein, and each separate value isincorporated into the specification as if it were individually recitedherein. Unless otherwise stated, all exact values provided herein arerepresentative of corresponding approximate values (e. g., all exactexemplary values provided with respect to a particular factor ormeasurement can be considered to also provide a correspondingapproximate measurement, modified by “about,” where appropriate). Allmethods described herein can be performed in any suitable order unlessotherwise indicated herein or otherwise clearly contradicted by context.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The description herein of any aspect or embodiment of the inventionusing terms such as reference to an element or elements is intended toprovide support for a similar aspect or embodiment of the invention that“consists of′,” “consists essentially of” or “substantially comprises”that particular element or elements, unless otherwise stated or clearlycontradicted by context (e.g., a composition described herein ascomprising a particular element should be understood as also describinga composition consisting of that element, unless otherwise stated orclearly contradicted by context).

This invention includes all modifications and equivalents of the subjectmatter recited in the aspects or claims presented herein to the maximumextent permitted by applicable law.

All publications and patent applications cited in this specification areherein incorporated by reference in their entireties as if eachindividual publication or patent application were specifically andindividually indicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to one of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1-52. (canceled)
 53. An isolated hetero-multimeric protein, which doesnot bind to CD16, and which comprises a first antigen-binding domain(ABD) that monovalently binds to a first antigen of interest and furthercomprises a second ABD which monovalently binds to a second antigen ofinterest, comprising: (a) a first polypeptide chain comprising a firstvariable domain (V) specific to the first antigen fused to a CH1 or CKconstant region, a second variable domain specific to the second antigenand comprised within the second ABD, and an Fc domain or portion thereofinterposed between the first and second variable domains; and (b) asecond polypeptide chain comprising a first variable domain (V) specificto the first antigen fused to a CH1 or CK constant region selected to becomplementary to the CH1 or CK constant region of the first polypeptidechain such that the first and second polypeptides form a CH1-CKheterodimer in which the first variable domain of the first polypeptidechain and the first variable domain of the second polypeptide form afirst ABD that binds the first antigen of interest, and wherein thefirst polypeptide chain comprises a third variable domain fused to thesecond variable domain, wherein the first and second polypeptide form aCH1-CK heterodimer, the first variable domain of the first polypeptidechain and the first variable domain of the second polypeptide chain forman antigen binding domain specific for the first antigen of interest,and the second and third variable domains of the first polypeptide chainform an scFv specific for the second antigen of interest, and stillfurther wherein wherein the hetero-multimeric polypeptide is a dimerwith a dimeric Fc domain, which dimeric Fc domain does not bind to CD16,and wherein said hetero-multimeric polypeptide has the domainarrangement: V1-(CH1 or CK)-Fc domain-V2-V3 V1-(CH1 or CK)-Fc domainwherein each V1, V2 and V3 is a heavy or light chain variable region;wherein the Fc domains comprise a CH2 and a CH3 domain capable ofCH3-CH3 dimerization; and further wherein one of the V1 of the firstpolypeptide chain and the V1 of the second polypeptide chain is a lightchain variable domain and the other is a heavy chain variable domain.54. An isolated hetero-multimeric protein, which does not bind to CD16,which comprises a first antigen-binding domain (ABD) that monovalentlybinds to a first antigen of interest and further comprises a second ABDwhich monovalently binds to a second antigen of interest, saidhetero-multimeric protein comprising: (a) a first polypeptide chaincomprising a first variable domain (V) specific to the first antigenfused to a CH1 or CK constant region, a second variable domain specificto the second antigen and comprised within the second ABD, and an Fcdomain or portion thereof interposed between the first and secondvariable domains which does not bind to CD16; and (b) a secondpolypeptide chain comprising a first variable domain (V) specific to thefirst antigen fused to a CH1 or CK constant region selected to becomplementary to the CH1 or CK constant region of the first polypeptidechain such that the first and second polypeptides form a CH1-CKheterodimer in which the first variable domain of the first polypeptidechain and the first variable domain of the second polypeptide form afirst ABD that binds the first antigen of interest, wherein thehetero-multimeric polypeptide is a trimeric polypeptide, comprising: (a)a first polypeptide chain comprising a first variable domain (V) fusedto a first CH1 or CK constant region, a second variable domain fused toa second CH1 or CK constant region, and an Fc domain or portion thereofinterposed between the first and second variable domains; (b) a secondpolypeptide chain comprising a variable domain fused at its C-terminusto a CH1 or CK constant region selected to be complementary to the firstCH1 or CK constant region of the first polypeptide chain such that thefirst and second polypeptides form a CH1-CK heterodimer; and (c) a thirdpolypeptide chain comprising a variable domain fused at its C-terminusto a CH1 or CK constant region, wherein the variable domain and theconstant region are selected to be complementary to the second variabledomain and second CH1 or CK constant region of the first polypeptidechain such that the first polypeptide chain and the third polypeptidechain form a CH1-CK heterodimer bound by disulfide bond(s) formedbetween the CH1 or CK constant region of the third polypeptide and thesecond CH1 or CK constant region of the first polypeptide, but notbetween the CH1 or CK constant region of the third polypeptide and thefirst CH1 or CK constant region of the first polypeptide such that thefirst, second and third polypeptides form a CH1-CK heterotrimer, andwherein the first variable domain of the first polypeptide chain and thevariable domain of the second polypeptide chain form an antigen bindingdomain specific for the first antigen of interest, and the secondvariable domain of the first polypeptide chain and the variable domainof the third polypeptide chain form an antigen binding domain specificfor the second antigen of interest; and further wherein the trimerichetero-multimeric polypeptide is a trimer with comprises a dimeric Fcdomain which does not bind CD16, which trimeric hetero-multimericpolypeptide has having the domain arrangement:

wherein each V-V pairing occurs between a light chain variable domainand a heavy chain variable domain, and wherein each constant regionpairing occurs between a CH1 and a CK.
 55. The isolated protein of claim53, wherein one of the first and second antigens of interest is anactivating receptor expressed by an effector cell and-the other antigenof interest is a cancer antigen.
 56. The isolated protein of claim 54,wherein one of the first and second antigens of interest is anactivating receptor expressed by an effector cell and-the other antigenof interest is a cancer antigen.
 57. A pharmaceutical compositioncomprising an isolated protein according to claim 53, and apharmaceutically acceptable carrier.
 58. A pharmaceutical compositioncomprising an isolated protein according to claim 54, and apharmaceutically acceptable carrier.
 59. A method of treating cancer ina subject comprising administering to the subject a compositioncomprising an isolated hetero-multimeric protein according to claim 53.60. A method of treating cancer in a subject comprising administering tothe subject a composition comprising an isolated hetero-multimericprotein according to claim
 54. 61. A method of making a heterodimericprotein, comprising: (a) providing a first nucleic acid encoding a firstpolypeptide chain according to claim 53; (b) providing a second nucleicacid encoding a second polypeptide chain according to claim 53; (c)expressing said first and second nucleic acids in a host cell to producea protein comprising said first and second polypeptide chains,respectively; and (d) loading the protein produced onto an affinitypurification support, optionally a Protein-A support, and recovering aheterodimeric protein.
 62. A method of making a heterotrimeric protein,comprising: (a) providing a first nucleic acid encoding a firstpolypeptide chain according to claim 54; (b) providing a second nucleicacid encoding a second polypeptide chain according to claim 54; (c)providing a third nucleic acid comprising a third polypeptide chainaccording to claim 54; (d) expressing said first, second and thirdnucleic acids in a host cell to produce a protein comprising said first,second and third polypeptide chains, respectively; and (e) loading theprotein produced onto an affinity purification support, optionally aProtein-A support, and recovering a heterotrimeric protein.
 63. Anexpression vector or vectors which comprise nucleic acids which incombination provide for the expression of a hetero-multimeric proteinaccording to claim 53, said expression vector or vectors comprising: (a)a first nucleic acid encoding a first polypeptide chain according toclaim 53; (b) a second nucleic acid encoding a second polypeptide chainaccording to claim 53; and (c) which first and second nucleic acids whenexpressed in combination in a host cell produce a hetero-multimericprotein according to claim
 53. 64. An expression vector or vectors whichcomprise nucleic acids which in combination provide for the expressionof a hetero-multimeric protein according to claim 54, said expressionvector or vectors comprising: (a) a first nucleic acid encoding a firstpolypeptide chain according to claim 54; (b) a second nucleic acidencoding a second polypeptide chain according to claim 54; (c) a thirdnucleic acid comprising a third polypeptide chain according to claim 54;and (d) which first, second and third nucleic acids when expressed incombination in a host cell produce a hetero-multimeric protein accordingto claim
 54. 65. An isolated or recombinant host cell which comprises anexpression vector or vectors according to claim
 63. 66. An isolated orrecombinant host cell which comprises an expression vector or vectorsaccording to claim 64.